Prevention of exercise-induced asthma by anatural isomer mixture of �-caroteneIttai Neuman, MD*; Hermona Nahum, MSc*; and Ami Ben-Amotz, PhD†

Background: The unicellular alga Dunaliella bardawil was previously shown tocontain very high concentrations of �-carotene composed of equal amounts of theall-trans and 9-cis stereoisomers which differ in their physicochemical features andantioxidative activity. Due to the controversy regarding the beneficial effect ofantioxidants on asthma, the acute effects of �-carotene of Dunaliella was assessedon airway hyperreactivity in patients with exercise-induced asthma (EIA).Methods: Thirty-eight patients with EIA participated in our study to verify the

antioxidative effect. The test was based on the following sequence: baseline pul-monary function, 7 minutes exercise session on a motorized treadmill, 8 minutesrest, 1-week oral random, double-blind supplementation of placebo or 64 mg/day�-carotene, pulmonary functions at rest, 7 minutes exercise session, 8 minutes restand again pulmonary functions.Results: All patients given placebo showed a significant postexercise reduction

of more than 15% in their forced expiratory volume in one second (FEV1). Of the38 patients who received a daily dose of 64 mg of �-carotene for 1 week, 20 (53%)were protected against EIA.Conclusions: Our results indicate that a daily dose of Dunaliella �-carotene

exerts a protective effect against EIA in some patients most probably through invivo antioxidative effect.

Ann Allergy Asthma Immunol 1999;82:549–553.

INTRODUCTIONStrenuous exercise may promote freeradical production, leading to lipid per-oxidation and tissue damage.1 There isevidence, however, that dietary supple-mentation with antioxidants, such asvitamin C and �-carotene, may protectagainst such damage.1,2 Studies on theuse of vitamin C in antigen inhalationchallenges,3 histamine inhalation4–7and methacholine inhalation tests4,8,9have yielded contradictory results.Proper assessment of the vitamin Ceffect on airways requires measure-ment of alterations in airway tone andairway obstruction, such as occurs af-

ter exercise-induced bronchospasm,10or in NO2-induced airway hyperre-sponsiveness.11 In asthmatic patients,pretreatment with ascorbic acid hasbeen shown to prevent the significantalterations in pulmonary functions in-duced by exercise.12 The present studywas undertaken in order to evaluate theprevention of exercise-induced asthma(EIA) by antioxidant supplement, suchas �-carotene. Dunaliella bardawil canaccumulate large amounts of �-caro-tene exceeding 10% of the algal dryweight.13–15 Natural �-carotene, asfound in D. bardawil, contains �50%all-trans �-carotene with the rest com-posed mostly of cis �-carotenes andminor amounts of a few other di-cis�-carotene isomers, which is slightlyhigher than the common one-third ciscomposition in fresh green vegetablesand fruits. Stereoisomers of carote-noids are distributed widely, but thepredominant forms of �-carotene are9-cis, 13-cis and 15-cis. Recently itwas suggested that 9-cis �-carotene

acts as an in vivo lipophilic antioxidantmore efficiently than does all-trans�-carotene.13 In the present study weexamined the effects of natural �-car-otene on hyper reactive airways at restand following the physical provocationof exercise.

METHODSA group of 38 patients with provenEIA participated in the study. Therewere 26 males and 12 females, rangingin age from 8 to 33 years (mean age16.3 years). All patients underwent aninitial evaluation of pulmonary func-tions in which all of them showed areduction of at least 15% in theirforced expiratory volume in 1 second(FEV1) after a 7-minute run on a mo-torized Quinton treadmill followed by8 minutes rest. The workload was thesame for each patient exercising up tosubmaximal effort as indicated by anOmeda 3700e pulse oxymeter. All sub-sequent exercise testing was performedunder the same ambient conditions forall patients, and with the same work-load that had produced EIA at thebaseline evaluation. Pulmonary func-tions were done at the same time of theday with similar ambient conditions toavoid any variability.Exercise testing as described above

was performed before and after 7 dayssupplementation period of 66 � 6 mgstereoisomer mixture of �-carotene orplacebo, administered randomly in adouble-blind fashion. Each patient un-derwent this protocol twice, receivingeither �-carotene or placebo, with 4weeks intervals between each protocolto allow washout. Twelve hours priorto evaluation of the pulmonary func-tions, all patients discontinued theirregular medications, which consistedof antihistamines, short-action bron-chodilators (salbutamol) and inhaledsteroids. The Helsinki Committee on

* Department of Allergy, Hasharon Hospital,Rabin Medical Center, Petach Tivka, Israel andSackler School of Medicine, Tel-Aviv Univer-sity, Tel-Aviv, Israel.† The National Institute of Oceanography, Is-

rael Oceanographic and Limnological Research,Haifa, Israel.Received for publication March 10, 1998.Accepted for publication in revised form De-

cember 14, 1998.

VOLUME 82, JUNE, 1999 549

the use of human approved the proto-col for this study. Informed consentwas obtained from each of the partici-pants.The �-carotene preparation used

was a dry powder of the �-carotenenaturally enriched alga Dunaliellabardawil. This organism containsmore than 6% of �-carotene on a dryweight basis,16–18 predominantly the9-cis stereoisomer of �-carotene. Thedried algae used contained 8% �-car-otene provided in hard capsules of 250mg powder or 22 � 1 mg �-caroteneper capsule (NBT, Nature Beta Tech-nologies Ltd., subsidiary of NikkenSohonsha Co, Gifu, Japan). Details onthe powder of D. bardawil, the cap-sules and the placebos have been de-scribed in detail elsewhere.13

Statistical AnalysisComparisons of means � SD betweenthe groups supplemented with eitheror Dunaliella �-carotene were madeby one-way analysis of variance(ANOVA). P �.05 was considered tobe significant. When statistical differ-

ences were found, we followed theanalysis with a Tukey standardizedrange test. T-tail probability was in-cluded.

RESULTSThirty-eight patients with EIA partici-pated in our study to verify the anti-oxidative effect. The test included thefollowing sequence: baseline pulmo-nary function, 7-minutes exercise ses-sion on a motorized treadmill, 8 min-utes rest, pulmonary function at rest,1-week oral random, double-blind sup-plementation of placebo or 64 mg/day�-carotene, pulmonary functions atrest, 7 minutes exercise session, 8 min-utes rest and again pulmonary func-tions. Each patient was treated consec-utively, at 4-week intervals, with either�-carotene or placebo and was tested 4times on each protocol for pulmonaryfunctions pre- and postexercise bothbefore and 7 days after taking the dailydose of the supplement. All the 38patients taking placebo revealed a sig-nificant postexercise reduction of more

than 15% in their FEV1. Twenty pa-tients out of the 38 (53%), which weresupplemented with a total dose of 448mg stereoisomer mixture of �-carotenein 7 days, showed a postexercise re-duction of less than 15% in FEV1 (Ta-ble 1).

DISCUSSIONOver 70% of asthmatic children expe-rience EIA19,20 while up to 98% of chil-dren who attend an asthma clinic com-plain of breathing discomfort uponexercising.21 The mechanism wherebythe airways respond to exercise is notyet fully understood, but probably in-volves bronchial muscle cells, mastcells and neurons22,23 with the releaseof mediators. Elevated levels of in-flammatory mediators have been dem-onstrated after exercise.24 Cyclo-oxygenase products of arachidonatemetabolism may also play a role in thepathogenesis of bronchial hyperreac-tivity.25,26 Vitamin C has been shownto shift the cyclooxygenase pathwayfrom synthesis of the bronchoconstric-

Table 1. FEV1 Values Before and After Exercise in EIA Patients Protected by Beta-Carotene

Patient Age

Placebo Treated Beta-Carotene Treated

Pre-Exercise Post-ExerciseChange %

Pre-Exercise Post-ExerciseChange %

Liter % of Predic. Liter % of Predic. Liter % of Predic. Liter % of Predic.

11 1.50 93 1.15 71 �23 1.82 113 1.71 106 �622 4.54 99 3.82 83 �16 4.47 97 4.10 89 �812 1.75 84 1.42 68 �19 1.68 81 1.76 85 �518 2.72 83 2.33 71 �14 2.70 83 2.48 76 �816 3.18 96 2.47 75 �22 3.37 102 3.00 91 �1117 4.84 104 3.06 66 �37 4.83 104 4.38 94 �917 3.69 98 2.84 75 �23 3.84 102 3.58 95 �715 4.10 106 2.97 77 �28 3.86 100 3.68 96 �514 2.18 70 1.81 58 �17 2.52 81 2.27 73 �1012 2.49 85 1.10 38 �56 2.15 73 2.11 72 �225 2.98 88 2.21 66 �26 2.81 83 2.50 76 �815 2.20 74 1.36 46 �37 2.11 71 2.30 77 �912 2.44 95 2.03 79 �17 2.30 89 2.25 87 �215 3.29 98 2.75 82 �16 3.23 96 3.18 94 �233 3.67 90 3.07 75 �16 3.89 95 3.67 90 �612 1.64 64 0.86 33 �50 1.99 77 1.91 74 �412 2.43 86 1.98 70 �19 2.18 77 1.88 66 �1414 3.53 93 2.57 68 �25 3.29 87 3.11 82 �512 2.12 87 1.63 67 �23 2.13 87 2.09 86 �222 3.57 87 2.90 71 �19 3.97 97 3.67 90 �8Mean �25.15 �5.15

� SD � 11.48 � 5.29(P �.05)

550 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY

tor PgF2 toward synthesis of the dilatorPgE2.27,28 It has been speculated thatduring exercise asthma free radicalsare produced to a higher extent by theexercising asthmatic and the effect ofthose free radical can be quenched bycertain antioxidants.29–38Kreisman et al,6 Mohsenin,11 and

Malo et al4 showed that the hydrophilicantioxidant vitamin C, has no signifi-cant effect on airway responsiveness atrest. When ingested as a single largedose (2 g) prior to exercise, vitamin Cprevented the development of EIA andreduced airway hyperreactivity to ex-ercise. These findings are in agreementwith those of Schachter andSchlesinger10 and Micirc and Hakhiu.12The mean level of ascorbic acid inasthmatic individuals is significantlylower than in non-asthmatic controls,29and physical, physiologic, emotionalor environmental stress does lowerplasma ascorbate levels.30 The etiologyof the increase in airway responsive-ness owing to exercise is not yet fullyunderstood, although inflammatorymediators and free radicals are thoughtto play a role in EIA. It is tempting tospeculate that some of the protectiveeffects of vitamin C may reside in itsantioxidant properties,27 its inhibitionof free radicals and mediators ofinflammation, and its shifting of thecyclooxygenase pathway from the syn-thesis of bronchoconstrictor to bron-chodilator prostaglandins.28During the present study, patients

were evaluated for exercise asthmapre- and postsupplementation with pla-cebo and �-carotene in attempt to ver-ify the antioxidative effect of the�-carotene against EIA. The majorityof our patients were protected by thesupplemented �-carotene. It is thereforetempting to assume that in patients withasthma �-carotene quenches the gener-ated increased amounts of reactive oxy-gen species from peripheral blood cellsand from cells recovered by bronchoal-veolar lavage.31 �-Carotene may reducethe pathophysiologic changes associatedwith asthma and its pathogenicity.32–35In patients with steroid-dependent

bronchial asthma, the free radical pres-ence is more intensive than in non-

steroid-dependent patients,36 and reac-tive oxygen species may induce anautonomic imbalance between themuscarinic receptor-mediated contrac-tion and the �-adrenergic-mediated re-laxation of the pulmonary smoothmuscle. Such autonomic imbalancemight be involved in the genesis ofbronchial hyperreactivity during lunginflammation.15 The reactive oxygenspecies induce bronchoconstriction, el-evate mucus secretion and cause mi-crovascular leakage.37,38 If reactive ox-ygen species participate in theinflammatory response occurring inairways disease, then antioxidantshould prove beneficial in therapy.38Indeed, ingestion of 1 g of vitamin Cor 30 mg of �-carotene diminished themarkers of lipid peroxidation at restand after exercise, but did not preventthe exercise-induced increase in oxida-tive stress.2 The amount of oxidativedamage depends on the exercise inten-sity and could be reduced through di-etary supplementation with antioxi-dants, such as vitamin C and E or�-carotene,1,39 all of which appear tobe very good quenchers of activatedforms of singlet oxygen and free radi-cals.40,41 So far, conventional treatmenthas not been able to correct either en-hanced lipid peroxidation or weak an-tioxidant defense.42Therapeutic action aimed at increas-

ing antioxidant defense mechanisms isstill a clinical challenge.43 Neverthe-less, bronchial asthma patients who re-ceived antioxidants in addition to con-ventional therapy were found toexhibit a more pronounced lowering ofchemiluminsecence in the blood, aswell as of the plasma malondialdehydecontent, than did patients who receivedconventional therapy alone.32,36 Withregard to the use of vitamin C to alle-viate asthma and to enhance athleticactivity, the available evidence is con-tradictory or inconclusive.44 For exam-ple, Malo et al4 found no significantchanges in FEV1 and forced vitalcapacity after ascorbic acid administra-tion as compared with placebo ad-ministration. It was concluded thatascorbic acid has no acute bronchodi-lator effect and does not alter bronchial

responsiveness in subjects with asth-ma.4,45,46 A deficiency of vitamin Cwas recorded in the majority of bron-chial asthma patients during exacerba-tion of their disease.27,47 Furthermore,pretreatment with ascorbic acid obvi-ated the significant alteration in airwaygeometry which is induced in asth-matic patients by exercise,12 and alsosignificantly mitigated the broncho-spasm normally observed 5 minutesafter exercise.10 It has been suggestedthat ascorbic acid exerts its effect byaltering arachidonic acid metabolism.27The results presented here on using

a stereoisomer mixture of Dunaliella�-carotene as an antioxidant are anextension of our previous studies inhumans and in animals.13–15 �-Caro-tene was administered to the subjectsin dry algal powder with no extraction,purification or modification in an at-tempt to verify the in vivo cellulareffect and antioxidative activity of9-cis �-carotene. It is assumed that theisomeric structure of �-carotene func-tions as an efficient quencher of singletoxygen and other free radicals, thuspreventing the consequent in vivo for-mation of dienic oxidized lipid prod-ucts.13 Nutritional supplementationthat contains both 9-cis and all-trans�-carotene will provide a synergisticprotection mechanism against damageby free radicals, initially by the oxida-tion of the labile 9-cis stereoisomer,and thereafter by the stable all-trans�-carotene. Stereoisomer mixture of�-carotene suppresses the attack ofcellular and reactive oxygen species,slows the formation of more radicals,and in turn avoids the destruction ofthe lipophilic parts of the cell or themembrane. Asthmatic subjects repre-sent a classical case study where thecellular level of free radicals is basi-cally high and increases upon strenu-ous exercise. A combination of differentantioxidants, hydrophilic and lipophilic,such as vitamin C and a mixture of ste-reoisomers of �-carotene, may undercertain conditions provide a better anti-oxidative effect than the use of onetype of quencher. Recently, the Alpha-Tocopherol, �-Carotene Cancer Pre-vention Study Group, CARET48 and

VOLUME 82, JUNE, 1999 551

the Physicians’ Health Study49 showedthat not only did synthetic all-trans�-carotene fail to reduce the incidenceof cancer and cardiovascular disease inhumans, but in fact it had an adverseeffect on the incidence of lung cancerand on the risk of death from lungcancer and cardiovascular disease.Experimental nutritional medical

studies with natural carotenoids, iso-mers of carotenes and carotenoidsoriginating from different plantsources specifically or synergisticallywith polar antioxidant have been lim-ited, and such research is in its infancy.The possible metabolic conversion of9-cis �-carotene to 9-cis retinol and9-cis retinoic acid was recently report-ed.50–52 The present study and recentlypublished studies13,53 show that evenwithin the scope of �-carotene itself,more attention should be paid to thestereoisomeric configuration and to themode of intake. We used dry algalpowder made from the whole plant thatcontains, aside from a high concentra-tion of �-carotene, other different mi-nor constituents common to greenplants. The possibility of synergisticeffects and the possible beneficial po-tency of the plant nutrients are stillobscure and warrant further research.In conclusion, the findings of the

present study clearly support the as-sumption that in some patients, dietarysupplementation with a stereoisomermixture of �-carotene, is protectiveagainst EIA. Physical activities arebeneficial for asthmatic patients, andingestion of certain antioxidants suchas vitamin C or �-carotene may enablethem to enjoy full participation in suchactivities.

REFERENCES1. Singh VN. A current perspective onnutrition and exercise. J Nutr 1992;122(Suppl)3:760–5.

2. Kanter MM, Nolte LA, Holloszy JO.Effects of an antioxidant vitamin mix-ture on lipid peroxidation at rest andpost-exercise. J Appl Physiol 1992;74:965–9.

3. Kordansky DW, Rosenthal RR, Nor-man PS. The effect of vitamin C onantigen-induced bronchospasm. J Al-lergy Clin Immunol 1979;63:61–4.

4. Malo JL, Cartier A, Pineau L, et al.Lack of acute effects of ascorbic acidon spirometry and airway responsive-ness to histamine in subjects withasthma. J Allergy Clin Immunol 1986;78:1153–8.

5. Cockcroft DW, Killian DN, MellonJJA, et al. Protective effect of drugs onhistamine-induced asthma. Thorax1977;32:429–37.

6. Kreisman H, Mitchell C, Bouhuys A.Inhibition of histamine-induced airwayconstriction: negative results withoxytriphylline and ascorbic acid. Lung1977;154:223–9.

7. Zuskin E, Lewis AJ, Bouhuys A. Inhi-bition of histamine-induced airwayconstriction by ascorbic acid. J AllergyClin Immunol 1973;51:218–26.

8. Ogilvy CS, Dubois AB, Douglas JS.Effects of ascorbic acid and indometh-acin on the airways of healthy malesubjects with and without inducedbronchoconstriction. J Allergy ClinImmunol 1981;67:363–9.

9. Mohsenin V, Dubois AB, Douglas JS.Effect of ascorbic acid on response tomethacholine challenge in asthmaticsubjects. Am Rev Respir Dis 1983;127:143–7.

10. Schachter EN, Schlesinger A. The at-tenuation of exercise-induced bron-chospasm by ascorbic acid. Ann Al-lergy 1982;49:146–51.

11. Mohsenin V. Effect of vitamin C onNO2-induced airway hyperresponsive-ness in normal subjects. Am Rev Re-spir Dis 1987;136:1408–11.

12. Micirc M, Hakhiu MA. Effect of vita-min C on exercise-induced broncho-spasm. Plucne Bolesti 1991;43:94–7.

13. Ben-Amotz A, Levy I. Bioavailabilityof a natural isomer mixture comparedwith synthetic all-trans �-carotene inhuman serum. Am J Clin Nutr 1996;63:729–34.

14. Bitterman N, Melamed Y, Ben-AmotzA. �-carotene and CNS oxygen toxic-ity in rats. J Appl Physiol 1994;76:1073–6.

15. Ben-Amotz A, Rachmilevich B,Greenberg S, et al. Natural �-caroteneand whole body irradiation in rats. RadEnviron Biophys 1998;37:187–93.

16. Ben-Amotz A, Katz A, Avron M. Ac-cumulation of �-carotene in halotoler-ant algae: Purification and character-ization of �-carotene rich globulesfrom Dunaliella bardawil (Chloro-phyceae). J Phycol 1987;18:529–37.

17. Ben-Amotz A, Lers A, Avron M. Ste-

reoisomers of �-carotene and phytoenein the alga Dunaliella bardawil. PlantPhysiol 1988;86:1986–91.

18. Avron M, Ben-Amotz A, Eds.Dunaliella: Physiology, biochemistry,and biotechnology. CRC Press Inc,1992:206–14.

19. Silverman M, Anderson SD. Standard-ization of exercise tests in asthmaticchildren. Arch Dis Child 1971;47:882–9.

20. Kattan M, Keens TG, Mellis CM, Le-vison H. The response to exercise innormal and asthmatic children. J Pedi-atr 1978;92:718–21.

21. Silverman M, Konig P, Godfrey S. Useof serial exercise tests to assess theefficacy and duration of action ofdrugs for asthma. Thorax 1973;28:574–8.

22. Pauwels R, Joos G, Van der StraetenM. Bronchial hyperresponsiveness isnot bronchial asthma. Clin Allergy1988;18:317–21.

23. Gaopp G. Exercise induced asthma.Pediatr Clin N Am 1975;22:63–76.

24. Lee TH, Nagakura T, Papageorgiou N,et al. Mediators in exercise inducedasthma. J Allergy Clin Immunol 1984;73:634–9.

25. O’Byrne PM. Airway inflammationand airway hyperresponsiveness.Chest 1986;90:575–7.

26. Fabbri LM, Aizawa H, O’Byrne PM,et al. An anti-inflammatory drug (BW755C) inhibits airway hyperrespon-siveness induced by ozone in dogs. JAllergy Clin Immunol 1985;76:162–6.

27. Rivers JM, Machlin LJ, eds. III Con-ference on vitamin C. Ann NY AcadSci 1987;498:229–47.

28. Bucca C, Rolla GA, Arossa W, et al.Effect of ascorbic acid on increasedbronchial responsiveness during upperairway infection. Respiration 1989;55:214–9.

29. Aderele WI, Ette SJ, Oduwole O, Ik-peme SJ. Plasma vitamin C (ascorbicacid) levels in asthmatic children. AfrJ Med Sci 1985;14:115–20.

30. Gleeson M, Robertson JD, MaughamRJ. Influence of exercise on ascorbicacid status in man. Clin Sci 1987;73:501–5.

31. Smith LJ, Houston M, Anderson J. In-creased levels of glutathione in bron-choalveolar lavage fluid from patientswith asthma. Am Rev Respir Dis 1993;147:1461–4.

32. Daniljak IG, Kogan AH, Bolijevic S.The generation of active forms of ox-

552 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY

ygen by the blood leukocytes, lipidperoxidation and antiperoxide protec-tion in bronchial asthma patients. TerArkh 1992;64:54–7.

33. Novak J, Nemeth I, Gyurkovitz K, etal. Examination of the role of oxygenfree radicals in bronchial asthma inchildhood. Clin Chim Acta 1991;201:247–51.

34. Bast A, Aaenen GR, Doelman CJ. Ox-idants and antioxidants: state of the art.Am J Med 1991;91(Suppl 3c):2S–13S.

35. De Lucia F, Bonavia M, Crimi E, et al.Anti-inflammatory-antioxidant treat-ment with a methane sulfonanilide inallergen-induced asthma. Ann Allergy1991;66:424–9.

36. Bolijevic S, Daniljak IG, Kogan AH.Changes in free radicals and possibil-ity of their correction in patients withbronchial asthma. Vojnosanit Pregl1993;50:3–18.

37. Doelman CJ, Bast A. Oxygen radicalsin lung pathology. Free Radic BiolMed 1990;9:381–400.

38. Barnes PJ. Reactive oxygen speciesand airway inflammation. Free RadicBiol Med 1990;9:235–43.

39. Witt EH, Reznick AZ, Viguie CA, etal. Exercise, oxidative damage and ef-fects of antioxidant manipulation. JNutr 1992;122(Suppl 3):766–73.

40. Ahmad S, Pardini RS. Mechanisms forregulating oxygen toxicity in phytoph-

agous insects. Free Radic Biol Med1990;8:401–13.

41. Sies H. Relationship between free rad-icals and vitamins: An overview. Int JVitam Nutr Res 1989;30(Suppl):215–23.

42. Abdusalamov AB, Shafter AM. Theefficacy of membrane-stabilizing ther-apy in bronchial asthma. Ter Arkh1994;66:32–4.

43. Leuenberger P. Respiratory diseasesand antioxidants. Schweiz MedWochenschr 1994;124:129–35.

44. Dvesen L. Vitamin therapy in the ab-sence of obvious deficiency. What isthe evidence? Drugs 1984;27:148–70.

45. Ting S, Mansfield LE, Yarbough J.Effects of ascorbic acid on pulmonaryfunctions in mild asthma. J Asthma1983;20:39–42.

46. Anderson R, Hay I, Van-Wik HA,Theron A. Ascorbic acid in bronchialasthma. S Afr Med J 1983;63:649–52.

47. Rozanov EM, Iutanova LK, Podorozh-nyi AP, et al. Vitamin PP and C allow-ances and their correction in the treat-ment of bronchial asthma patients.Vopr Pitan 1987;6:21–4.

48. Omenn GS, Goodman GE, ThornquistMD, et al. Effects of a combination ofbeta carotene and vitamin A on lungcancer and cardiovascular disease.New Eng J Med 1996;334:1150–5.

49. Hennekens CH, Buring JE, Manson

JE, et al. Lack of effect of long-termsupplementation with beta carotene onthe incidence of malignant neoplasmand cardiovascular disease. New EnglJ Med 1996;334:1145–9.

50. H�buterne X, Wang XD, Johnson EJ,et al. Intestinal absorption and metab-olism of 9-cis �-carotene in vivo: bio-synthesis of 9-cis retinoic acid. J LipidRes 1995;36:1264–73.

51. Nagao A, Olson JE. Enzymatic forma-tion of 9-cis, 13-cis, and all-trans reti-nals from isomers of �-carotene.FASEB J 1994;8:968–73.

52. Wang XD, Krinsky NI, Benotti PN,Russell RM. Biosynthesis of 9-cis reti-noic acid from 9-cis �-carotene in hu-man intestinal mucosa in vitro. ArchBiochem Biophys 1994;313:150–5.

53. Gaziano JM, Johnson EJ, Russell RM,et al. Discrimination in absorption ortransport of �-carotene isomers afteroral supplementation with either all-trans or 9-cis �-carotene. Am J ClinNutr 1995;61:1248–52.

Request for reprints should be addressed to:A Ben-AmotzIsrael Oceanographic and LimnologicalResearch,

P.O.B. 8030, Haifa 31080Israelemail: ami@ocean.org.il

VOLUME 82, JUNE, 1999 553