Historical aspects of the major neurological vitamin ...

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Chapter 29

Historical aspects of the major neurological vitamin

deficiency disorders: overview and fat-soluble vitamin A

DOUGLAS J. LANSKA*

Veterans Affairs Medical Center, Tomah, Wisconsin, and University of Wisconsin School of Medicine andPublic Health, Madison, Wisconsin, USA

VITAMINSANDDIETARYDEFICIENCY DISEASES

Introduction

Vitamins are organic micronutrients that are essentialto normal growth and metabolism, and are presentin only minute amounts in natural foodstuffs. Histori-cally, vitamin deficiency disorders have been majorcauses of neurological morbidity and mortalitythroughout the world, often affecting large segmentsof malnourished populations. The neurological presen-tations vary with the deficiencies involved, butincluded dementia, amnestic confabulatory states,delirium, acute psychosis, blindness, eye movementabnormalities, ataxia, myelopathy, polyneuropathy,and congenital neural tube defects.

Although diseases that are now recognizable as vita-min deficiency disorders have been known for millen-nia, the “vitamin doctrine” was not developed untilthe early 20th century (Hopkins, 1929/1965). Prior tothis development, vitamin deficiency disorders weregenerally attributed to toxic or infectious causes, themost powerful pathophysiological paradigms of thelate-19th and early-20th centuries.

Since the initial development of the vitamin doc-trine, there has been an explosive growth in our under-standing of cellular metabolism, including thecoenzyme functions of many of the vitamins. In somecases we now possess a fairly complete pathophysiolo-gical understanding of the development of specificneurological vitamin deficiency disorders, are able toidentify such disorders early, are able to treat andoften cure people with such disorders when identifiedearly using synthetic forms of the vitamins, and most

importantly are able to prevent these disorders in thefirst place by targeted supplementation and foodfortification. As a result, incidence, prevalence, casefatality, and mortality of neurological vitamindeficiency disorders have declined dramatically indeveloped countries since the middle of the 20th cen-tury. Endemic forms of these disorders have beeneither eliminated from or greatly curtailed indeveloped countries, and the relatively rare residualcases generally reflect individual predispositionsbecause of altered intake (e.g., alcoholics, food fad-dists, total parenteral nutrition), malabsorption (e.g.,pernicious anemia, chronic diarrhea, iatrogenic causes,alcoholism), and altered metabolism or abnormal utili-zation.(e.g., medications, coincident disease). Unfortu-nately, high rates of many neurological vitamindeficiency disorders persist in developing countriesand other populations besought with war, famine,and poverty.

This chapter begins a historical review of vitamindeficiency disorders causing neurological illness,and includes a general overview of the historicalorigins of the vitamin doctrine and an historical reviewof fat-soluble vitamin A. Study of the history ofneurological vitamin deficiency disorders can berewarding from several vantage points, and can aug-ment understanding of normal neurochemistry andneurophysiology, neuropathophysiology, the interrela-tionships between neurological and systemic illness,neurotherapeutics, neuroepidemiology, and neurologi-cally-oriented social medicine and public health.Seldom in the case of neurological disorders is such abreadth and depth of medical understanding availableto help prevention and treatment efforts.

*Correspondence to: Douglas J. Lanska MD, VA Medical Center, 500 E Veterans St., Tomah, WI 54660, USA.

E-mail: [email protected], Tel: +1-608-372-1772, Fax: +1-608-372-1240.

Handbook of Clinical Neurology, Vol. 95 (3rd series)History of NeurologyS. Finger, F. Boller, K.L. Tyler, Editors# 2009 Elsevier B.V. All rights reserved


Components of a physiologicallycomplete diet and Hopkins’“accessory food factors”

In the late-19th century, a physiologically complete dietwas believed to require only a sufficient amount ofproteins, carbohydrates, fats, inorganic salts, andwater. However, as early as 1880 and 1881, in studiesfor a doctoral thesis, Russian physician Nicolai I. Lunin(1853–1937) in Dorpat (now Tartu, Estonia) found thatmice did not thrive or grow when fed on purified dietscontaining only these constituents, although he madeno effort at identifying any missing essential factorsin the inadequate diets (Hopkins, 1929/1965; Voss,1956; Rosenfeld, 1997).

In 1905, Cornelius Pekelharing (1848–1922) inUtrecht performed similar experiments and achievedsimilar results, but found that animals that receivedmilk instead of water thrived. Pekelharing suggestedthat there was an unrecognized substance in milk,present in very small amounts, which was necessaryfor the animals to adequately utilize the other dietarycomponents—a clear statement of what would laterbe called the “vitamin doctrine” (Hopkins, 1929/1965;Rosenfeld, 1997).

Unfortunately, the early reports by Lunin, Pekelhar-ing, and others attracted little attention until the workof British biochemist Frederick Hopkins (1861–1947)at Cambridge University was published in 1912. From1906 to 1912 Hopkins conducted similar feeding experi-ments with young mice and rats (Hopkins, 1929/1965).Hopkins’ experiments were notable for providing care-ful observations under controlled laboratory conditionswith “analytical control of materials, for the meticu-lous weighing and measurement of the quantities offood consumed, for the many days over which animalswere studied . . ., for consistent recording of the weightand growth of the rats, and for . . . independence fromcurrent dogma” (Weatherall, 1990, p. 123). Hopkinsfound that rats fed purified mixtures of protein(casein) or amino acids, carbohydrates (sucrose,starch), fats (butter or lard), mineral salts, and waterfailed to grow or even lost weight and died, unlessthe diet was supplemented with small amounts of milk(Hopkins, 1906, 1912, 1929/1965). Hopkins concludedthat milk contained “accessory food factors” that arerequired in trace amounts for normal growth (Hopkins,1912, 1929/1965). Although there were clearly otherswho anticipated this work, and although other investi-gators had difficulty reproducing and confirmingHopkins’ results, Hopkins shared the 1929 Nobel Prizein Physiology or Medicine for his contributions tothe “discovery of the growth-stimulating vitamins”(Hopkins, 1929/1965).

From Funk’s “vitamine” to vitamin

In 1911 and 1912 Polish chemist Casimir Funk (1884–1967), then working at the Lister Institute for Preven-tive Medicine in London, proposed that the active diet-ary factor that was effective in the treatment of animalmodels of beriberi was a specific organic substancepresent in trace amounts—one of several trace dietaryfactors that were essential for life and which, whendeficient, resulted in such diseases as beriberi, scurvy,rickets, and pellagra (Funk, 1911, 1912, 1922; Harrow,1955). Funk had isolated a concentrate from rice polish-ings that seemed to be curative for polyneuritis inpigeons, and that his chemical analyses suggested wasprobably an amine. Because this substance appearedto be vital for life, Funk named it “vitamine” for “vitalamine” (Funk, 1912).

Funk supposed that vitamines would all belong tothe same chemical class, just as amino acids as the con-stituents of proteins are chemically related. However,Funk’s concentrates were primarily nicotinic acid,which was contaminated with small amounts of theanti-beriberi factor (thiamin). Nevertheless, Funk’sterm was widely adopted and applied to a series offood substances, regardless of their chemical struc-tures. Indeed, the introduction of the term and itspopularization led to further research efforts interna-tionally, as these dietary substances became recognizedas clinically important beyond the prevention of somedistant tropical diseases. In 1920, Jack Drummond(1891–1952) proposed that the “e” be dropped from“vitamine,” because there was no evidence that theessential dietary factors are amines; the revisedterm “vitamin” would then conform to a standardnomenclature convention, one in which substances ofundefined composition end with the suffix “-in”(Drummond, 1920; Rosenfeld, 1997).

VITAMINA DEFICIENCY: NIGHTBLINDNESSANDKERATOMALACIA

Introduction

Vitamin A is integral to sensory transduction and spe-cifically the transduction of light for visual perception.Vitamin A is the precursor of the visual pigmentswithin the rods and cones of the retina. In particular,a derivative of vitamin A, 11-cis retinal, is the chromo-phore within the G protein-coupled photoreceptor pro-tein, rhodopsin, which is localized to the outersegments of rod cells in the retina. This transmem-brane detector undergoes a conformational change inresponse to light, which activates an intracellularG-protein-coupled transduction cascade, and ultimatelycellular responses that lead to visual perception. The

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study of this physiological process has not only greatlyexpanded knowledge of sensory transduction, visualperception, and dark adaptation, but has provided aprototypical model and tremendous insights intothe functions of a large class of structurally relatedG protein-coupled cell receptors involved, for example,in detecting odorants, neurotransmitters, and hor-mones (Hargrave and McDowell, 1992; Filmore, 2004).

Because rhodopsin is available in greater quantitiesthan any other G protein-coupled receptor, it has beenstudied in much greater detail than its fellow G pro-tein-linked receptors (Hargrave and McDowell, 1992).Drugs targeting members of this integral membraneprotein family now represent nearly half of all pre-scription pharmaceuticals and are a major focus ofcurrent drug development (Filmore, 2004).

The neurological disorder associated with vitamin Adeficiency is night blindness, which has plagued mal-nourished populations for millennia, and remains amajor public health problem in many countries aroundthe world.

Description of night blindnessand keratomalacia

Night blindness was recognized by the ancient Egyptiansand the ancient Greeks (Dowling and Wald, 1958; Wolf,1996). During the Roman Era, Galen (ca. 129–199 A.D.)clearly described “nyktalopia” as night blindness andrecommended eating raw beef liver (now known to con-tain high concentrations of vitamin A) to correct thecondition (Wolf, 1996). Chinese medical texts alsodescribed night blindness: Sun-szu-mo (7th centuryAD) recommended pig liver as a treatment (Wolf,1996). Later, during European colonial expansion,several physicians—including Jacobus Bontius (1592–1631), the first European physician in the Dutch EastIndies, and Willem Piso (1611–1678) in Brazil—describednight blindness and its cure with shark liver (Wolf,1996). In 1754, German physician C. A. von Bergendescribed epidemics of night blindness in rural Russiaand postulated a nutritional cause (Wolf, 1996). Englishphysician William Heberden (1710–1801), often consid-ered the outstanding clinician of his era, also describeda case of “nyctalopia, or night-blindness” in a sailorwho developed blindness at night, with retained daytimevision, which gradually abated within 3 days of goingashore (Heberden, 1818, pp. 269–270).

The nutritional basis of night blindness was furtherelaborated by Austrian naval physician EduardSchwarz (1831–1862) during an around-the-worldscientific expedition (1857–1859) (Wolf, 1996). Nightblindness (“hemeralopia”) developed in 75 of the 352men on board, often simultaneously with scurvy. Every

evening at dusk, afflicted sailors became blind and hadto be led about the ship. Several affected sailors,after being kept below decks for 3–5 days, transientlyregained normal night vision, but became night blindagain when exposed to sunlight (although mysteriousat the time, this no doubt resulted from marginalvitamin A reserves, which could be tenuously renewedwith minimal light exposure, but which were insuffi-cient for more rapid rates of depletion). Schwarzfound that both boiled ox liver and pig liver werecurative.

Corneal epithelial defects were clearly recognized inthe 19th century in those subsisting on diets now recog-nizable as deficient in vitamin A. Corneal ulcerationwas reported in 1817 by Francois Magendie (1783–1855) among vitamin A-deficient dogs fed for severalweeks on a diet limited to sugar and water, althoughhe erroneously attributed this to a deficiency of dietarynitrogen (i.e., protein) (Budd, 1842; Wolf, 1996). In1842, English physician George Budd (1808–1882)described similar corneal ulcers among East Indiansduring a sea voyage to England (Budd, 1842; Hughes,1973; Wolf, 1996), and in 1857 African explorer DavidLivingstone reported corneal lesions among Africannatives who subsisted on coffee, manioc, and meal(Wolf, 1996). In 1904, Masamichi Mori in Japandescribed widespread xerophthalmia (corneal dryness),often with keratomalacia (ulceration and perforationof the cornea), among Japanese children subsistingmostly on rice and barley, and further reported thatliver and cod-liver oil were curative (Mori, 1904; Wolf,1996).

The association between night blindness with cor-neal epithelial defects was recognized in the late-19thand early-20th centuries. In 1860, V. von Hubbenetfirst reported the association between night blindnessand corneal epithelial defects (“silver scales on the cor-nea”), attributed this to an inadequate diet, and foundit treatable with beef liver (Wolf, 1996). In 1862, Bitotreported foamy white spots (“Bitot spots”) on thecorneas of children with night blindness (Wolf, 1996),and by 1863 concluded that night blindness andxerophthalmia are both manifestations of the samedisorder (Bitot, 1863; Wolf, 1996, 2001). Similarly, in1913, S. Ishihara recognized the association of nightblindness and keratomalacia in malnourished children(Ishihara, 1913; Wolf, 1996).

In 1919, during World War I, E. Bloch studiedmalnourished Danish children with night blindnessand keratomalacia, who had subsisted on fat-free milk,oatmeal, and barley soup (Bloch, 1919; Wolf, 1996).In a critical experiment, Bloch prospectively studied32 institutionalized toddlers (aged 1–4 years), half ofwhom received animal fat (whole milk and butter),

Au1 HISTORICAL ASPECTS OF THE MAJOR NEUROLOGICAL VITAMIN DEFICIENCY DISORDERS 437


while the others received vegetable fat (margarine).The animal fat group remained healthy, whereas 50%of the vegetable fat group developed corneal xerosis(Bloch, 1919; Wolf, 1996, 2001). All of the xerosiscases were rapidly cured with cod-liver oil. Blochconcluded that whole milk, butter, and cod-liver oilcontain a fat-soluble substance that protects againstxerophthalmia.

Boll’s discovery of the visual pigment

In 1876 and 1877, Franz Boll (c1849–1879) observed arelationship between retinal color and light exposure:(1) the frog retina is paler after light exposure andcan become completely colorless in direct sunlight;and (2) excised animal retinas that had been exposedto light are colorless, but the purple color is restoredif animals are kept in the dark for a period of timeafter exposure to light before they are killed (Boll,1876, 1877, 1877/1977;Baumann, 1977; Hubbard, 1977;Kuhne, 1879/1977; Wolf, 2001). Boll concluded thatlight causes bleaching of the retinal pigment, and alsosuggested that the outer segments of the rods containa substance that conveys an impression of light to thebrain by a photochemical process.

Kuhne’s experiments with retinalpreparations and rhodopsin

Willy Kuhne (1837–1900), professor of physiology at theUniversity of Heidelberg, stimulated by Boll’s observa-tions, began experimental studies of the retina in 1877,and continued these over a productive 5-year period,resulting in a series of 22 important papers (;;;Kuhne,;Au2

Ewald and Kuhne, 1877; Crescitelli, 1977). By the timehe began his retinal studies, Kuhne was already arenowned physiologist who had previously isolated andnamed the digestive protease trypsin from the pancreas,coined the word “enzyme,” and contributed greatly tothe biochemistry of protein digestion (Wolf, 2001).Kuhne pursued his retinal studies with similar zeal andwith arguably even more important results.

In frogs, Kuhne confirmed that visual purple (Sehpur-pur) was bleached by light, but maintained its color in thedark, even after death. In a darkroom illuminated by redlight, Kuhne perfected a technique for isolating frogretinas, which remained purple in the dark but becamecolorless when exposed to sunlight (Kuhne, 1879/1977;Wolf, 2001). In contrast to previous opinions (includingBoll’s) that the retinal pigment is red, Kuhnewas adamantthat the rod pigment is in fact purple and named it visualpurple. This bleaching process progressed through severaldifferent color stages (from purple to orange to yellow tobuff and then to colorless), which Kuhne correctly inter-preted as indicating chemical transformations, because

of the changing absorption spectra and the changingfluorescence of the different stages in ultraviolet light(Kuhne, 1879/1977; Wolf, 2001). It is now known that rho-dopsin (11-cis-retinal plus the protein opsin) is purple withblue fluorescence, an intermediate stage is orange withcontributions of purple and yellow, all-trans-retinal-opsinis yellow, and the end-product of the light-bleachingprocess, free all-trans-retinol, is colorless with greenfluorescence (Wolf, 2001).

The rate of photo-bleaching is dependent on tempera-ture, as well as on the intensity and wavelength of light.Kuhne correctly concluded that photo-bleaching is aphotochemical process, and not a strictly thermal pro-cess, because infrared light is invisible and does notbleach the retinal preparations.

The photo-bleaching process was also found to bereversible and dependent of the retinal pigment epithe-lium (Kuhne, 1879/1977; Wolf, 2001). An excised frogeyeball could be fully bleached after being kept in sun-light for 30 min, but the purple color still reappears inthe dark, independent of the circulation of the blood.In contrast, an isolated, bleached retinal preparationseparated from the retinal pigment epithelium isunable to regenerate the purple color. However, if theisolated bleached retinal preparation is placed onto anisolated retinal pigment epithelium, the purple colordoes regenerate, demonstrating unequivocally theessential role of the retinal pigment epithelium inpigment regeneration within the retina.

The process of regeneration of the visual pigmentwas puzzling though, as it was “extraordinarily slow”(too slow by far to account for the rapidity of changingvisual sensation), and because it seemed to occur bytwo processes: one slow process different than a sim-ple reverse of the bleaching process (which Kuhnenamed neogenesis), and a relatively rapid process thatcould reverse the intermediaries (but not the finalproduct) back to visual purple (which Kuhne namedanagenesis).

Kuhne localized visual purple (rhodopsin) to theouter segments of the rods within “platelets” (nowcalled “disks”), and observed that bile or bile salts dis-solve the rods, bringing rhodopsin into solution whereit could be further studied chemically (Kuhne, 1879/1977; Wolf, 2001). Kuhne further surmised that rho-dopsin included a protein moiety, because it was alarge molecule that, when in solution, did not diffusethrough a semipermeable membrane and could be pre-cipitated with ammonium sulfate (Kuhne, 1879/1977;Wolf, 2001).

As early as 1877, Kuhne likened vision to a repeti-tive photographic process (Wald, 1950). Kuhne devel-oped this idea further when he found he was able tosee images bleached onto the retina. After having a

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frog stare into a flame for 14 h, he isolated its retinaand observed a bleached area in the shape of aninverted flame. Kuhne found he could create other ret-inal images, which he called “optograms,” after havingfrogs or rabbits stare at a window for several minutes.Kuhne’s optograms stimulated widespread speculationthat such images could be used forensically to deter-mine the guilty party from the retinal image depictingsomeone just murdered. Kuhne initially dismissed suchspeculation; however, by 1880, when a young man wasbeheaded by guillotine in the nearby town of Bruschal,Kuhne apparently had a different viewpoint and imme-diately retrieved the corpse, extracted the eyes in adimly lit room screened with red and yellow glass,and within 10 min of the decapitation viewed one ofthe few reported human optograms (Wald, 1950).

Kuhne’s work concluded with his proposed “opto-chemical hypothesis,” which attributed vision to aphotochemical change in visual purple (rhodopsin),such that the chemical products or some processrelated to the chemical change is responsible for stimu-lating the visual cells and thereby conveying a visualimage. Kuhne’s prescient concept of photochemicaltransduction would later be shown to be largely cor-rect, particularly with the important work of Waldand colleagues beginning in the 1930s.

McCollum, Osborne, and Mendel, andthe discovery of fat-soluble vitamin A

In 1913, Elmer Verner McCollum, PhD (1879–1967) atthe University of Wisconsin in Madison, with hisvolunteer assistant Marguerite Davis, discovered afat-soluble accessory food factor, present only in cer-tain fats and distinct from the water-soluble anti-beri-beri factor (McCollum and Davis, 1913, 1914;McCollum, 1964; Day, 1997; University of WisconsinCollege of Agricultural and Life Sciences). Rats fedon a diet of presumably pure protein (casein), carbohy-drates, and salt grew well for several months, but thenstabilized or lost weight, unless the diet was supple-mented with certain “lipins” that were extractable withether and were present in butter fat and eggs. McCol-lum felt that the lapse of growth after several monthswas due to the exhaustion of body stores of someunrecognized organic growth factor that was normallypresent in certain fats. Similar work was presentedalmost simultaneously by Lafayette Mendel (1872–1935) of the Sheffield Scientific School (affiliated withYale University) and Thomas Osborne (1859–1929) ofthe Connecticut Agricultural Station in New Haven,who together also reported the high potency of codliver oil in supporting growth under these conditions(Osborne and Mendel, 1913, 1914).

In 1913, Gowland Hopkins and A. Neville suggestedthat both American groups had been able to obtaingrowth with the diets of casein and lactose, becausethese substances had been incompletely purified andwere contaminated with a water-soluble growth factor(Hopkins and Neville, 1913). In further studies, McCol-lum and Simmonds (1916) confirmed Hopkins’ suspi-cion and concluded that rats need both a water-soluble factor and a fat-soluble factor for growth. In1916, McCollum and his graduate student CorneliaKennedy labeled these “fat-soluble A” and “water-soluble B” (McCollum and Kennedy, 1916; Day, 1997).

McCollum initially believed that “fat-soluble A” was asingle vitamin capable of treating both xerophthalmia andrickets (McCollum and Kennedy, 1916); however, in 1922,McCollum and colleagues demonstrated that cod-liver oilcould be treated (by aeration at 100�C for at least 12 h) soas to eliminate its efficacy against xerophthalmia, whilemaintaining its antirachitic activity in rats (McCollumet al., 1922). Ultimately the anti-xerophthalmia factorwas named vitamin A and the anti-rachitic factor wasnamed vitamin D.

Linking visual manifestationsto vitamin A deficiency

In 1913, Ishihara proposed that a “fatty substance” inblood is necessary for synthesis of both rhodopsinand the surface layer of the cornea, and that nightblindness and keratomalacia develop when this sub-stance is deficient. Shortly thereafter, Osborne andMendel showed that, in the absence of the dietary sup-plementation with certain fats, rats developed weightloss, night blindness, and corneal ulcers, thus illustrat-ing the most important physiological functions of vita-min A (i.e., support of vision, epithelial differentiation,and growth), and also providing an experimental modelof human night blindness and keratomalacia (Osborneand Mendel, 1914; Wolf, 1996). In 1925, Fridericia andHolm directly linked vitamin A to night blindness inanimal experiments: vitamin A-deficient rats, whenlight adapted (i.e., with light-“bleached” retinas) andplaced in the dark, formed rhodopsin at a slower ratethan did normal rats (Fridericia and Holm, 1925; Wolf,1996). In 1929, Holm demonstrated the presence ofvitamin A in retinal tissue.

In the late 1930s, Wald and colleagues began studiesof experimentally induced human vitamin A deficiencyand demonstrated a progressive rise in the visualthreshold over a month-long period on a vitaminA-deficient diet, and subsequent rapid resolution ofthe deficit over 90 min upon ingestion of carotene(i.e., provitamin A) (Wald and Steven, 1939). A numberof subsequent studies in animals and man further

HISTORICAL ASPECTS OF THE MAJOR NEUROLOGICAL VITAMIN DEFICIENCY DISORDERS 439


corroborated the association between vitamin A defi-ciency and night blindness and keratomalacia (Dowlingand Wald, 1958; Wolf, 2002).

The isolation, chemical structure, andchemical synthesis of vitamin A

In attempts to isolate the active growth-promoting fac-tor in fats, Osborne and Mendel (1914) obtained anactive yellow oil from butter, egg yolks, and cod-liveroil, but not from lard or olive oil. Steenbock (1919) sub-sequently noted that active growth-promoting extractsfrom butter, egg yolk, or carrots are yellow, whileactive extracts from liver or kidney are white. In1920, Steenbock and Gross suggested that fat-solublefactor A is associated with a yellow pigment and isconverted in vivo to an active colorless form (Steen-bock and Gross, 1920; Wolf, 1996). However, aroundthe same time, Palmer and Kempster (1919) fed chick-ens a diet free of yellow pigments (i.e., white corn,skimmed milk, bone meal, and small amounts of porkliver) and discovered that the chickens neverthelessgrew normally and laid eggs that (despite colorlessegg yolks) produced normal chicks.

These confusing results were not resolved until 1930,when Moore, in experiments with rats, showed that theactive yellow pigment extracted from plants, butterfat, or egg yolks (b-carotene) is a precursor (i.e., provi-tamin) that is indeed converted to an active colorlessfactor (vitamin A or retinol) in vivo and accumulateswithin the liver (Moore, 1930; Wolf, 1996). Later studiesfound that the enzymatic conversion of b-carotene toretinol occurs in the intestinal mucosa (Wolf, 1996).

In the 1930s, Paul Karrer (1889–1971) and colleaguesat the University of Zurich isolated b-carotene (themain dietary precursor of vitamin A) and retinol, anddetermined their chemical structures (Karrer et al.,1931; Karrer, 1937/1966). Karrer shared the 1937 NobelPrize in Physiology or Medicine “for his investigationson carotenoids, flavins and vitamins A and B2” (Kar-rer, 1937/1966). In 1947, Isler and colleagues completedthe full chemical synthesis of vitamin A. The availabil-ity of vitamin A through food fortification and medic-inal supplements virtually eliminated ocular vitamin Adeficiency from developed countries by the second halfof the 20th century (Underwood, 2004).

Wald and the visual cycle of vitamin A

After completing his graduate studies in zoology withSelig Hecht (1892–1947) at Columbia University (Wald,1948b, 1967/1972; Dowling, 2000, 2002), American phy-siologist George Wald (1906–1997) obtained a NationalResearch Council Fellowship in Biology (1932–1934) to

pursue the photochemistry of vision. Within the spaceof a single Wanderjahre, Wald worked in three differ-ent laboratories under the guidance of three interna-tionally recognized mentors, all of who ultimatelyreceived the Nobel Prize in Physiology or Medicinefor different contributions (Wald, 1935a,b, 1967/1972;Dowling, 2000, 2002).

Wald began work in the laboratory of biochemistOtto Warburg (1883–1970) at the Kaiser-Wilhelm-Insti-tut (now the Max-Planck-Institut) fur Biologie in Ber-lin-Dahlem, Germany, where he dissected animalretinas to obtain the light-sensitive compound rhodop-sin (Dowling, 2002). Based on a chemical test and theabsorption spectrum, Wald tentatively concluded thatthe retina contains vitamin A, a finding that he subse-quently confirmed in the laboratory of Paul Karrer(1889–1971) at the University of Zurich (Warburg hadsuggested the transfer because Karrer had elucidatedthe structure of vitamin A in 1931) (Karrer, 1937/1966). After this, Wald moved briefly to the cellularmetabolism laboratory of Otto Meyerhof (1884–1951)at the Kaiser Wilhelm Institute for Medical Researchin Heidelberg, Germany, where Wald discovered thevisual cycle of vitamin A (Wald, 1935a,b; Wolf, 2001).

Wald’s former mentor Hecht had proposed, based onthe relationship between photosensitivity and time duringdark adaptation, that dark adaptation “follows the courseof a bimolecular reaction. . .[and that] visual reception indim light is conditioned by a reversible photochemicalreaction involving a photosensitive substance and its twoproducts of decomposition” (Hecht, 1919, pp. 516–517).Wald confirmedHecht’s prediction and showed that visualpurple (rhodopsin) is decomposed by light into a com-pound he called “retinene” and a protein (later called“opsin”) (Wald, 1935a,b; Karrer, 1937/1966; Wolf, 1996).Retinene was subsequently shown by R. A. Morton andT. W. Goodwin to be the aldehyde of vitamin A, “retinal-dehyde,” now known as “retinal” (Morton and Goodwin,1944; Ball et al., 1946). Retinal could either recombine withopsin to reform rhodopsin, or could instead be convertedto retinol (vitamin A). Because “vitamin A is the precursorof visual purple [rhodopsin], as well as the product of itsdecomposition,”Wald proposed that the “visual processestherefore constitute a cycle” (Wald, 1935b, p. 368).

During the 1930s, Wald also initiated studies ofcone vision and was able to extract a red-sensitive pig-ment from chicken retinas that he called iodopsin(Dowling, 2000; Kresge et al., 2005). In the mid-1950s, Wald and colleagues showed that iodopsinbleaches to form retinal and a protein, which is differ-ent from the protein opsin in rhodopsin. Hence, Waldproposed the terms “scotopsin” for the opsin of rods,and “photopsins” for the opsins of cones (Wald et al.,1955b; Dowling. 2000).

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Wald’s group subsequently elaborated the enzymaticconversions of various elements in the rhodopsin system(Wald, 1948a; Wald and Hubbard, 1949, 1950; Wald andBrown, 1950; Hubbard and Wald, 1951; Hubbard, 1956;Dowling, 2000). By the mid-1950s, Wald, Ruth Hubbard(Wald’s secondwife), and Paul Brown,with the assistanceof several organic chemists, determined that the rhodop-sin system is dependent upon an isomerization of retinal(a conformational change in the molecule), and specifi-cally that the 11-cis isomer of retinal was the precursorof all visual pigments (Wald and Hubbard, 1950, 1955aAu3 ;Hubbard and Wald, 1952; Hubbard et al., 1953; Brownand Wald, 1956; Hubbard, 1956, 1966; Oroshnik et al.,1956). 11-cis retinal is twisted and sterically hindered andstable only in the dark. Light causes isomerization to theall-trans form of retinal, which in turn must eventuallybe re-isomerized for the visual cycle to continue (Hubbardand Kropf, 1958; Hubbard, 1966). By slowing the chemi-cal processes with liquid nitrogen, Wald’s group alsodemonstrated that rhodopsin goes through a series ofvery transient molecular transformations, and that oneof these intermediaries (meta-rhodopsin II) triggers exci-tation of the photoreceptor before retinal is ultimatelyhydrolyzed from opsin (Matthews et al., 1963).

In 1942, Hecht and colleagues had demonstrated thata single photon might be enough to trigger excitation ina rod. In 1965, Wald suggested that a large chemicalamplification must occur for a single photon to be ableto trigger such excitation of the rod, and by analogywith the blood clotting system this amplification couldpotentially occur by a cascade of enzymatic reactions.Later studies showed that rhodopsin is a transmembraneprotein consisting of seven membrane-spanning helices,that are interconnected by extracellular and intracellularloops, and that form a binding pocket for its ligand, 11-cis retinal. Absorption of light by 11-cis retinal causesisomerization to all-trans retinal and propagation of aconformational change in the protein to the cytoplasmicsurface. The meta-rhodopsin II intermediary then inter-acts with transducin, a G-protein, to activate phospho-diesterases that control cyclic GMP levels, which inturn modulate release of neurotransmitter from therod cell (Stryer, 1986; Hargrave and McDowell, 1992).

In 1967, Wald was recognized with a Nobel Prize inPhysiology or Medicine for “discoveries concerning theprimary physiological and chemical visual processes inthe eye” (Wald, 1967/1972).

Hormone-like actions of vitamin A

In 1960, Dowling and Wald showed that almost all ofthe non-visual roles of vitamin A are carried out byretinoic acid, and further that retinoic acid could notbe metabolized to retinol (the form in which vitamin

A is transported), or to retinyl esters (the form inwhich vitamin A is stored), or to retinal (the aldehydeneeded for synthesis of visual pigments) (Dowlingand Wald, 1960). As a result, their rats maintainedwith retinoic acid (but not vitamin A or its variousprovitamins, i.e., dietary carotenoids) grew normallybut became extremely night blind and eventually per-manently blind. As rhodopsin concentrations declined,visual thresholds rose, followed by loss of opsin, disin-tegration of the outer segments of the rods, and severedropout of visual cells. Because retinoic acid is notstored in the body and cannot be metabolized to a sto-rage form, the animals stopped growing within a fewdays of deprivation of retinoic acid and developedsevere systemic symptoms within 1–2 weeks.

Subsequently retinoic acid was found to act in ahormone-like fashion to regulate gene expression(Ross and Ternus, 1993), a role for vitamin A thatWolf and De Luca had proposed as early as 1970(Wolf and De Luca, 1970; Wolf, 1996). In 1987, P.Chambon and colleagues in Strasbourg, France, andR. M. Evans and colleagues in San Diego, simulta-neously discovered retinoic acid receptors in cellnuclei, which bind with retinoic acid to modulate geneexpression, thereby influencing embryonic develop-ment, cellular differentiation (including that of thecornea), and growth (Giguere et al., 1987; Chambon,1996; Wolf, 1996).

Public health interventions toaddress vitamin A deficiency in

developing countries

The first global survey of xerophthalmia conducted bythe World Health Organization in the early 1960s sug-gested a significant prevalence of the disorder in manycountries (Ooman et al., 1964), but was later found tohave seriously underestimated the global burden ofvitamin A deficiency (Sommer et al., 1981; Reddy,2002; Underwood, 2004). Subsequently, various inter-vention trials were conducted, including controlledtrials in Jordan and India in the late 1960s, whichdemonstrated the feasibility of periodic vitamin A dos-ing to prevent vitamin A deficiency disorders (Reddy,2002). In 1975, under the auspices of the US Agencyfor International Development (USAID) and the WorldHealth Organization (WHO), the International VitaminA Consultative Group was established at a meeting ofthe United Nations International Children’s EmergencyFund (UNICEF) in New York, to coordinate and facili-tate activities to combat vitamin A deficiency disordersworldwide (Reddy, 2002; Underwood, 2004).

By the 1980s, global estimates suggested 13 millionchildren suffered from xerophthalmia and another



40–80 million children were at risk (Sommer et al.,1981; Underwood, 2004). But within 20 years these esti-mates were again found to underestimate the burdenof illness resulting from vitamin A deficiency, withrecent estimates suggesting that 3 million preschoolchildren have clinical signs of vitamin A deficiencyannually, and that another 140–250 million preschoolchildren are at risk based on serum vitamin A levels(Underwood, 2004).

In the 1980s and 1990s, large randomized, double-blind, placebo-controlled clinical trials were conductedin developing countries. They demonstrated that vita-min A supplementation could reduce childhood mortal-ity by approximately one quarter to one third, even bygiving concentrated vitamin A supplements at 6-monthintervals (Sommer et al., 1986; Beaton et al., 1993;Semba, 1999; Underwood, 2004).

Vitamin A supplementation of malnourished chil-dren is now considered one of the most cost-effectivehealth interventions known (World Bank, 1993; Semba,1999). Although consistent periodic distribution of vita-min A supplements can control vitamin A deficiencydisorders, sustained control by this means is fragile,especially in countries with economic decline and civilunrest (Underwood, 1999Au4 , 2004). As was the case witheradication of endemic pellagra in the United States inthe early-20th century (Lanska, 2002), other measuresare also needed to ensure adequate diets and improvesocio-economic conditions before vitamin A deficiencydisorders can be truly controlled (Underwood, 1998,2004; Underwood and Smitasiri, 1999).

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