Synnøve Liaaen-Jensena, Bjørn Bjerkengb and Marianne
Østerliec
a Organic Chemistry Laboratories, Norwegian University of
Science and Technology,
N-7491 Trondheim,
Norway. E-mail: slje@chembio.ntnu.no
b AKVAFORSK, Institute of Aquaculture Research
AS, N-6600 Sunndalsøra, Norway.
c Department of Food Science, HiST, N-7004 Trondheim, Norway.
[Bek-Nielsen Foundation Lecture presented
at the OFIC2000 Conference, 4th Sept. 2000, K.L.]
Abstract. The terms optical isomerism (chirality)
and geometrical isomerism (trans-cis or E/Z) are illustrated. trans-cis Isomerism of carotenoids is treated
in a general way, and natural carotenoid cis-isomers
are exemplified in conjunction with important biological processes. cis-Carotenoids are the most common
isolation artefacts and procedures for proof of natural occurrence are cited.
Controlled trans-cis photochemical isomerization using iodine or diphenyl diselenide
as catalyst results in quasi-equilibrium mixtures considered to reflect
thermodynamic equilibrium. Recommended methods for modern analysis of trans- and cis-isomers are treated. The paper focuses on selective absorption
and deposition of carotenoid cis-trans isomers in various animal tissues,
and biological functions as possible provitamin A, antioxidants, in gap
junction communication etc., in relation to nutrition and health. Relevant
evidence is discussed for three selected carotenoids, namely for astaxanthin
including work by the authors, and comprehensive recent literature data for
lycopene and b-carotene.
The apparent digestibility coefficients
(ADCs) and selective accumulation of cis-trans
isomers of astaxanthin in plasma and various tissues of some fish species are
reported. In decreasing order the following ADCs have been found in rainbow
trout: all-trans-astaxanthin > 13-cis-astaxanthin > 9-cis-astaxanthin. The fish species
investigated selectively accumulate all-trans-astaxanthin
in plasma and muscle (salmonid fishes) and 13-cis-astaxanthin in the liver. In contrast, astaxanthin cis-isomers accumulate selectively in
human plasma. A mixture of astaxanthin cis-trans-isomers is apparently a better in vitro antioxidant than all-trans-astaxanthin.
All-trans
lycopene is considered as the major
isomer in fresh tomato, the major dietary source of lycopene. Nevertheless more
than 50% of total lycopene is present as cis-isomers
in human serum and an even larger proportion of cis-isomers in prostate tissue. Higher bioavailability of cis-lycopene and/or in vivo isomerization are suggested. Lycopene has no provitamin A
activity, but is an efficient singlet oxygen quencher and an effective
antioxidant. Critical evaluations on epidemiological and intervention studies
involving lycopene are available.
All-trans
b-carotene is the superior provitamin A,
whereas the 9-cis isomer has around
25% relative activity. In most natural sources all-transb-carotene is
the dominant isomer, including human plasma. Green Dunaliella algae produce around 40% of total b-carotene as the 9-cis isomer. In vivo
isomerization of 9-cis to all-trans-b-carotene has been documented after its oral administration
to humans prior to plasma absorption. b-Carotene is considered as an unusual type of antioxidant
associated with certain health beneficial effects. Clinical tests with b-carotene supplements are referred to.
In conclusion, species dependant
selective biological effects of trans-
and cis-configurated carotenoids in
animal systems are documented in recent years, and the effects on nutrition and
health aspects including cancers and coronary diseases deserve further
attention.
[Key
words: Carotenoids, cis-trans isomerism, nutrition health,
astaxanthin, lycopene, b-carotene.]
Carotenoids are usually yellow-red isoprenoid polyene pigments widespread in Nature. They are biosynthesized de novo by all photosynthetic organisms, certain bacteria and fungi. Animals may selectively absorb carotenoids from the diet, and may also have the ability to modify selected carotenoids structurally by metabolism1.
Structures have been assigned to some 700 naturally occurring carotenoids.2,3 Variations in the common C40-skeleton are encountered by e.g. C50-carotenoids or apocarotenoids with shorter carbon skeletons. Several different functional groups may be present in the xanthophylls (oxygen containing carotenoids) and include allene and epoxide, as exemplified by neoxanthin (1, Fig. 1), acetylene, butenolide, glycoside, sulphate etc.2,3
CHIRALITY (OPTICAL ISOMERISM)
Stereogenic or chiral centres in carotenoids are generally carbon atoms with four different substituents, marked with an asterisk for 1. Each chiral centre may have two alternative chiralities referred to as R or S by the Cahn-Ingold convention,4 see Fig. 1. For neoxanthin (1) the trisubstituted allenic group represents a chiral axis with two alternative absolute configurations. Chiral carotenoids exhibit optical activity as demonstrated by CD (circular dichroism) spectra.5 In this paper optical isomerism is not of major concern.
Figure 1. Optical and Geometrical Isomerism in Carotenoids: Neoxanthin (1)
Geometrical isomerism is generally referred to as cis-trans isomerism or E/Z isomerism by more recent, unequivocal nomenclature based on the sequence rules.4 The trans/cis and E/Z designations are illustrated in Fig. 1. Trans and E are usually synonymous for carotenoids unless oxygen substituents are present in the polyene chain. The model 1 is presented with all-trans configuration.
In principle each double bond in the polyene chain may exist in the cis-configuration. However, some cis-bonds (7- and 7’-cis, 11- and 11’- cis, see numbering on 2 in Fig. 2) are sterically hindered and unfavoured. In common dicyclic carotenoids cis-configuration is generally encountered in 9 (9’), 13 (13’) or 15-position.
A cis double bond results in bent molecules as illustrated for the 5-cis, 9-cis, 13-cis and 15-cis isomers of lycopene (2) in
Fig. 2.
Figure 2. All-trans-Lycopene
(2) and its cis-Isomers
In principle several mono-cis and poly-cis-isomers are possible. However, in practice the number of cis-isomers is reduced by steric and electronic factors. Mono-cis-isomers are most frequently encountered. cis-Isomers in general crystallize or aggregate less readily than the all-trans isomer. Higher solubility in lipophilic solution may facilitate transport within cells and between tissues. Moreover, the bent shapes are likely to effect their incorporation into lipoproteins or interaction with proteins.
Natural cis-isomers
Trans to cis isomerization is a facile process that takes place readily in organic solvents. cis-Isomers consequently represent the most common isolation artefacts.6 Refined isolation procedures including fast extraction in darkness at low temperature and subsequent HPLC analysis are required in order to establish the natural occurrence of cis-isomers.7
Most natural carotenoids have all-trans configuration. However, various mono-cis isomers are naturally occurring and prolycopene is a poly-cis (7,9,7’,9’-tetra-cis)-isomer with two sterically hindered cis-bonds.8 In some cases the cis-configuration has an established biological significance. Thus 9-cis-neoxanthin (1), not its trans-isomer, is the biological precursor of cis-configurated abscissic acid which is a plant growth regulator.9 Moreover, 15-cis carotenoids are functional in the photosynthetic reaction centre of phototropic bacteria10,11 and of green plants,10 and 15-cis-phytoene is the biosynthetic C40-precursor of coloured all-trans carotenoids1 with various functions. The 11-cis to trans isomerization of retinal which is derived from b-carotene (3) is an important step in the visual process.12 Information is accumulating on the presence of cis/trans mixtures of carotenoids in bacteria living under stressed conditions. Reference is made to strongly isomerized bacterioruberin in halophilic bacteria exposed to strong sunlight13 and to psycrophilic bacteria in Arctic regions.14
Iodine catalyzed isomerization in light in benzene or dichloromethane solution has been a standard procedure for obtaining reproducible cis/trans stereoisomeric mixtures.15 Recently, we have successfully employed diphenyl diselenide as a promotor for photo-chemical stereoisomerization.16 Closely similar quasi-equilibrium mixtures of cis/trans stereoisomers are obtained with the two catalysts, suggesting that thermodynamic equilibrium is reached. For most carotenoids the all-trans isomer predominates in the equilibrium mixture with lesser amounts of various mono-cis isomers and a small amount of di-cis isomers. cis- Bonds adjacent to keto groups appear not to be favoured, judged by our results for fucoxanthin.17 Certain structural features in the polyene chain favour cis-configuration, i.e. acetylenic bonds or in-chain oxidized methyl groups. In such cases adjacent cis-bonds are strongly favoured.18-20 No sterically hindered cis-bonds are formed during the isomerization, and 15-cis isomers are generally only obtained in minor amounts. Most cis-isomers can be reversibly isomerized to the same equilibrium mixture obtained from the trans-isomer. Naturally occurring or synthetic, sterically hindered cis-isomers are irreversibly isomerized.21
Analysis of trans- and cis-isomers
Chromatographic separation
of geometrical isomers is best achieved by HPLC (high performance liquid
chromatography), preferably by instruments supplied with a diode array detector
that allows simultaneous recording of VIS (visible light) spectra. Reversible
isomerization before recording the diagnostically important VIS spectrum is
thereby avoided. Several chromatographic systems have been developed.2
Both normal phase and reversed phase systems are employed. HPLC is used both
for analytical and preparative separations and is indispensable for work with
geometrical isomers of carotenoids.
The VIS spectra of cis-isomers exhibit characteristic changes relative to the all-trans isomer, including hypsochromic shift, reduced spectral fine-structure and the appearance of a so-called cis-peak.15 cis-Isomers generally have lower extinction coefficients than the all-trans isomer. Only in a few cases, mainly for synthetic, crystallized cis-carotenoids22 have the real extinction coefficients for carotenoid-cis-isomers been determined and approximate values are used in quantitative studies. 1H NMR is the method of choice for definite assignment of the position of a cis double bond in carotenoids.5 Characteristic down-field shifts are observed for protons in the vicinity of the cis double bond. Certain chiral carotenoids exhibit so-called conservative CD spectra.23 For such carotenoids the Cotton effect becomes opposite if a cis-bond is introduced.5
In conclusion, the identification of carotenoid cis-isomers can be performed reliably on the microgram scale by combination of the modern techniques available. If the same extinction coefficient is used for the all-trans and various cis-isomers present in a stereoisomeric mixture, the proportion of cis-isomers is underestimated because the extinction coefficient of cis-isomers is generally lower than for the all-trans isomer.
Relevant evidence will be
discussed for three selected carotenoids, namely astaxanthin (4, Fig. 3), lycopene (2, Fig. 2) and b-carotene (3, Fig. 3). Included are results from
the authors’ laboratories and relevant recent literature data.
Figure 3. Structures of b-Carotene, Retinol and
Astaxanthin
Astaxanthin (4, Fig. 3) is biosynthesized de novo by some bacteria,25 microalgae,26 yeast27 and higher plants,28 and is the major carotenoid of many seafood species including wild salmonid fishes and crustaceans.29 The commercially most important sources of astaxanthin are chemically synthesized products, the main part of which is used in diets for aquacultured species. Due to the manufacturing process the products contain partly isomerized astaxanthin, whereof the cis-isomers represent approximately 25%. Reviews covering numerous papers on carotenoids of seafoods and aquacultured species in general30 and on pigmentation of salmonid fishes in particular31,32 reveal that carotenoid cis/trans isomerism has been largely ignored. However, we recently addressed the digestion and accumulation of astaxanthin cis/trans isomers in some fish species and humans. The results are reviewed briefly here.
In the first experiment we investigated the apparent digestibility coefficients (ADC), muscle accumulation,33 and the relative concentrations in plasma, liver, skin, and kidney of astaxanthin cis/trans-isomers34 in rainbow trout (Oncorhynchus mykiss). The fish were fed diets containing either predominantly all-trans-astaxanthin (97% of total astaxanthin) or a mixture of all-trans and cis-astaxanthins (64 and 36%, respectively). The cis/trans isomeric mixture of astaxanthin was produced from crystalline astaxanthin by an iodine-catalyzed isomerization. ADC of total astaxanthin was significantly higher in trout fed all-trans-astaxanthin (79%) compared to trout fed the cis/trans mixture (64%). In decreasing order the following ADCs were found: all-trans-astaxanthin > 13-cis-astaxanthin > 9-cis-astaxanthin. Accordingly, a higher carotenoid concentration was observed in plasma of trout fed diets with all-trans-astaxanthin compared to trout fed the astaxanthin stereoisomeric mixture, and muscle carotenoid concentration tended to be higher (10.0 and 8.6 mg kg-1, respectively). The relative cis/trans-isomer concentrations of muscle, intestinal tissues, plasma, skin, and kidney were not affected, whereas all-trans-astaxanthin was higher in intestinal tissues and 13-cis-astaxanthin was lower in liver of trout fed all-trans-astaxanthin. Regardless of diet, the relative concentrations of astaxanthin cis-isomers in muscle and plasma were ca. 5 and 7% of total astaxanthin, respectively. However, the relative amount of 13-cis-astaxanthin was elevated pronouncedly in the liver (39-49% of total astaxanthin).
In a comparative trial with astaxanthin (the astaxanthin source consisted of 75% all-trans-, 3% 9-cis- and 22% 13-cis-astaxanthin) in a diet for Atlantic salmon (Salmo salar) and Atlantic halibut (Hippoglossus hippoglossus) a higher digestibility of astaxanthin was observed in the latter species.35 Atlantic halibut does not deposit carotenoids in the muscle, but small amounts were found in plasma and liver. As with rainbow trout, low levels of astaxanthin cis-isomers were present in the plasma and muscle (salmon), whereas elevated levels of 13-cis-astaxanthin were found in the liver (20-32% of total astaxanthin). In Arctic charr (Salvelinus alpinus) fed a diet with cis-astaxanthins comprising 25% of total astaxanthin, low levels of cis-astaxanthins (ca. 5% of total astaxanthin) were detected in the muscle.36
Other workers37
failed to detect astaxanthin in the blood of humans given a salmon meal
containing canthaxanthin and astaxanthin, and concluded that astaxanthin was
not absorbed by the intestine. In contrast, we found maximum plasma levels of
ca. 1.3 mg/L after 7 hrs following ingestion of a meal containing 100 mg
astaxanthin consisting of 74% all-trans-,
9% 9-cis- and 17% 13-cis-astaxanthin.38 13-cis-Astaxanthin accumulated selectively,
and the distribution of astaxanthin geometrical isomers was different from that
of the meal: 49.4 % for all-trans-astaxanthin,
13.2% for 9-cis- astaxanthin, and
37.4 % for 13-cis-astaxanthin. Our
results indicate that a selective process increases the relative proportion of
astaxanthin cis-isomers compared to
the all-trans-astaxanthin during
blood uptake. The results show that
geometrical isomers of astaxanthin are distributed selectively in different
tissues of fish. Furthermore, blood uptake of astaxanthin cis/trans isomers appears
to be governed by different mechanisms in different animals. Astaxanthin
is metabolized into the colourless 3-hydroxy-4-oxo-b-ionone and its reduced form 3-hydroxy-4-oxo-7,8-dihydro-b-ionone by rat hepatocytes,39 and
provided the fish is not saturated with vitamin A into vitamin A1
and A2 in salmonid fishes.40 Selective metabolic
conversion of the different isomers may account for our observations.
Astaxanthin has strong antioxidant properties.41 We have compared the antioxidant properties of all-trans-astaxanthin with a mixture of astaxanthin cis/trans isomers in cod liver oil and fatty acid ethyl esters enriched in eicosapentaenoic acid and docosahexaenoic acid.42 The mixture of astaxanthin cis/trans isomers appeared to be the better antioxidant. Astaxanthin is essential to growth of Atlantic salmon (Salmo salar) alevins.43 Dietary intake of the astaxanthin-producing yeast (Xanthophyllomyces dendrorhous) or synthetic astaxanthin improves health status of rainbow trout, possibly by suppressing the formation of lipid peroxides.44,45 Antitumor activity is reported in mice fed astaxanthin.46,47 These properties may be related to the antioxidative actions of astaxanthin.
Lycopene (2, Fig. 2) is an aliphatic carotene without terminal rings. The major dietary sources are tomato and tomato products such as paste, ketchup, soup, juice, etc. Less concentrated sources are various fruits including watermelon and pink grapefruit.48,49 Synthetic lycopene and tomato-based lycopene are now commercially available. Lycopene is used for coloring food products such as yogurt and fruit juices.49 Absorption from the diet and deposition in specific biological tissues are documented. Thus lycopene is encountered in various human tissues including plasma, testis, adrenal gland, liver, lung, kidney and prostate.48,50-53 In rats administered [14C] lycopene, labeled lycopene was detected in liver, stomach, intestine, colon, pancreas, kidney, lung and ovary.54
In vitro, lycopene (2) readily undergoes trans to cis isomerization, cf. Fig. 2. Some fourteen different mono-, dicis- and tricis- isomers have been prepared and fully characterized.55 In tomato all-trans lycopene was long considered to be the major geometrical isomer,48 where it occurs together with the previously overlooked 5-cis isomer which exhibits the same VIS absorption spectrum and no cis-peak. A 45:55 ratio of the all-trans and 5-cis isomer in Nature has recently been quoted.49 Processing by cooking results in breaking the vegetable matrix56,57 and in cis-isomerization and results in higher bioavailability.58,59 In ferrets cis-lycopenes were more bioavailable than the trans-isomer.60 Around ten different geometrical isomers of lycopene have been detected in human serum, including the 5-cis61, 9-cis, 13-cis and 15-cis isomers depicted in Fig.2;51,61-64 58-37% of total lycopene are cis-isomers.52 An even greater proportion, 79-88% of total lycopene, was found as cis-isomers in prostate tissues.52,65 Since several cis-isomers were detected in plasma of humans fed a diet supplemented with tomato puree devoid of these isomers,64 an in vivo trans-cis isomerization might be implied.
Lycopene has no vitamin A activity, but has received much attention in recent years in a health perspective.52,56,62 No evidence for toxicity by high consumption of lycopene has been reported. Extensive reviews on the biology and implications of lycopene for human health and disease are available.48,53,61,65 Critical evaluations on epidemiological and intervention studies involving lycopene, and effects on cancers of the digestive tract, cervix, breast, skin, bladder and prostate, as well as on cardiovascular disease are given.53,65 Studies with animal models and limited work on cell cultures are listed.53,65 A recent review of the epidemiological literature regarding tomato based products and blood lycopene levels versus cancer, concluded that the benefit was strongest for cancers of the prostate, lung and stomach.66,67 However, the need for further fundamental studies on the biology of lycopene, including absorption, metabolism, excretion and function in experimental models and humans has been pointed out.53,65 Concerning the metabolism of lycopene, 5,6-dihydroxy-5,6-dihydrolycopene is a major metabolite in human plasma68 and the presumed metabolites epimeric 2,6-cyclolycopene-1,5-diols with five-membered rings have been detected in breast milk and serum of lactating women.69
The chemistry of the antioxidant action of lycopene is receiving great interest. Lycopene is an effective singlet oxygen quencher65,70 and an effective antioxidant.71 Under conditions of free-radical initiated autoxidation of carotenoids the sequence lycopene > b-carotene > zeaxanthin > lutein was observed.72 Gap junction communication between cells is stimulated by lycopene and some other carotenoids.48,52,73,74
Health benefits of lycopene may be related to the above properties. However, specific studies serving to differentiate biological effects or functions of all-trans and various cis-isomers abundant in human tissues are required.
b-Carotene
b-Carotene (3) or b,b-carotene by IUPAC nomenclature2, is the most widely distributed and abundant carotene in Nature. Carrot is a rich dietary source, and b-carotene is present in most vegetables and fruits.
The all-trans isomer is by far the dominant one in natural sources. In palm oil derived from Elaeis guineensis and E. olifera only around 1% cis-b-carotene of total b-carotene has been reported.75 Even a carotene concentrate from crude palm oil had a cis : trans ratio 2 : 98 for b-carotene.76 Synthetic all-trans b-carotene is produced on an industrial scale and is widely used for food and feed additives as a combined provitamin A and colorant. Commercial, formulated products are likely to contain some 9-cis and 13-cis isomers. The green alga Dunaliella bardawil produces under stressed conditions, involving high light intensities and nitrogen deficiency, the 9-cis isomer in around 40% of total b-carotene.72 A parallel biosynthetic pathway for cis-carotenes has been claimed, but not proved1. Isomeric b-carotene ex Dunaliella is marketed as a health product.
All-trans-b-carotene (3) is the superior provitamin A and provides retinol (5, vitamin A) upon metabolic central or excentric cleavage,1,78,79 Fig. 3. Vitamin A is essential for mammals, and the corresponding aldehyde, retinal, is involved in the visual process.12 9-cis-b-Carotene is a less efficient provitamin A. An efficiency of around 25% for the neo U (9-cis) isomer relative to all-trans-b-carotene has been reported for rats.80 The metabolic fate of the 9-cis-b-carotene is a subject of recent interest, since 9-cis-retinoic acid apparently can be formed in vitro and in vivo.81 In contrast to the several cis-lycopenes encountered in human serum only all-trans-b-carotene has been detected in serum when an isomeric mixture also containing the 9-cis isomer is ingested.82-84 Other workers report low proportions of 9-cis-b-carotene in serum under similar conditions,85-87 as well as in breast milk and buccal mucosa.81,87 In vivo 9-cis to all-trans isomerization of b-carotene has been suggested86 and documented in studies88 in which 13C-b-carotene was orally administered to humans before plasma absorption. 13-cis-b-Carotene was detected in amounts up to 10% of total b-carotene in serum of patients suffering from erythropoietic protoporphyria after ingestion of synthetic b-carotene.89
Besides its important functions as a provitamin A b-carotene has been characterized as an unusual type of antioxidant.90 Thus b-carotene was found in in vitro experiments to exhibit good radical trapping and antioxidant behavior at low oxygen pressure, corresponding to the low oxygen partial pressures in most tissues under biological conditions. At higher oxygen pressures b-carotene loses its antioxidant behavior and shows an autocatalytic, prooxidant effect.90 In a model study no significant difference in the rate of oxidative degradation for 9-cis and all-trans b-carotene was found.91 However, other workers have reported a higher antioxidant activity of the 9-cis b-carotene compared to the all-trans isomer in vitro92. In vivo effects in rats were interpreted as higher antioxidative effect of 9-cis-b-carotene compared with that of the all-trans isomer, judged by enhanced degradation of the 9-cis isomer in the livers.93
Provitamin A and antioxidant functions have been associated with certain health benefits of b-carotene. Also the ability of b-carotene to quench singlet oxygen94 may have a bearing on its biological effects. It remains to be learned if singlet oxygen occurs biologically other than in photosensitized reactions.95
Epidemiological and intervention studies for humans, comprising four large-scale clinical trials, testing the effect of supplementary b-carotene on the risk for chronic diseases including cancers and coronary heart disease, have been carried out in recent years. These trials were generally based mainly on all-trans b-carotene supplements . Authoritative and critical reviews are available.96-98 These and other papers99-101 discuss possible mechanisms and functions. A discussion of this material is outside the scope of this paper.
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