Vitamine—vitamin. The early years of discovery

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Clinical Chemistry 43: 680-685, 1997;

Clinical Chemistry. 1997;43:680-685.)

© 1997 American Association for Clinical Chemistry, Inc.

 

Articles

Vitamine—vitamin. The early years of discovery

Louis Rosenfeld

 

New York University Medical Center, Department of Pathology, 560 First

Ave., New York, NY 10016.

 

 

Abstract

 

In 1905, Cornelius Adrianus Pekelharing found that animals fed

purified proteins, carbohydrates, fats, inorganic salts, and water

would thrive only if small amounts of milk were added to the diet. He

concluded that the milk contained some unrecognized substance that in

very small quantities was necessary for normal growth and maintenance.

In 1911, Casimir Funk isolated a concentrate from rice polishings that

cured polyneuritis in pigeons. He named the concentrate " vitamine "

because it appeared to be vital to life and because it was probably an

amine. Although the concentrate and other " accessory food substances "

were not amines, the name stuck, but the final " e " was dropped. In

1913 two groups discovered a " fat-soluble " accessory food substance.

Initially believed to be a single vitamin, two separate factors were

involved. One, effective against xerophthalmia, was named vitamin A;

the other, effective against rickets, was named vitamin D. The factor

that prevented scurvy was isolated in 1928. Known as " water-soluble

C, " it was renamed ascorbic acid.

 

 

Key Words: indexing terms: accessory food substance • nutritional

deficiency • indispensable dietary constituent

 

Introduction

 

For many years, diseases had been vaguely known to result from some

dietary deficiencies. Near the start of the 20th century, students of

nutrition were investigating not deficiency diseases as such, but what

were the components of a physiologically complete diet. They believed

that a well-balanced diet need contain only a suitable amount of

proteins, carbohydrates, fats, inorganic salts, and water. Advances in

chemistry had made it possible to prepare a large number of these

substances (proximate principles) as chemical compounds, and many

investigations were undertaken to determine the quality and optimum

amounts of these ingredients in an " average daily diet. " There was a

wide range from which to choose. However, animals fed these highly

purified foodstuffs did not thrive or grow.

 

The earliest such study was by N. Lunin in Gustav von Bunge's

(1844–1920) Laboratory in Basle (1881). He reported that young mice

did not thrive on an artificial mixture of the purified components of

milk (proteins, fats, carbohydrates, and salts) and consequently, that

this synthetic milk diet lacked " unknown substances " without which

life could not be sustained. This work was not followed up, attracted

little attention, and was forgotten. Orthodox opinion preferred a

simpler explanation for the nutritional failure of the experimental

animals: The purified diets were so unpalatable and monotonous that

loss of appetite, malnourishment, and death were inevitable.

 

In 1905, Cornelius Adrianus Pekelharing (1848–1922) of Utrecht carried

out similar experiments with mice and purified foodstuffs, and got

results similar to those of Lunin. If milk was given instead of water,

the mice thrived upon the diet. He concluded that an unrecognized

substance was present in milk, which, even in very small quantities,

is important for nutrition, and without which the animal loses the

ability to utilize the other components of its diet. The report,

hidden in a Dutch medical journal, did not become widely known.

 

In 1884, efforts were being made to eliminate beriberi from the

Japanese navy by giving the sailors increased amounts of meat, barley,

and fruit. These dietary reforms were introduced by Surgeon General

Kanehiro Takaki (1849–1915) and resulted in the eradication of

beriberi from the navy. Clinical signs of beriberi primarily involve

the nervous system, e.g., muscular atrophy and peripheral paralysis.

Takaki correctly attributed the disease to a food deficiency, but

mistakenly believed that sufficient amounts of protein prevented it.

 

Christiaan Eijkman (1858–1930; Fig. 1 ), a Dutch

physician-physiologist, provided an impetus for further investigation

working in the Dutch East Indies (Indonesia). In 1897, he discovered

that the disease known as polyneuritis in animals and beriberi in

humans could be induced in chickens and pigeons by a diet restricted

to polished rice. The birds are unable to fly, walk, or even to stand.

Cure and prevention was achieved by feeding them the unpolished rice

or the rice polishings. For many years most medical authorities,

influenced by the work of Pasteur, believed that a bacterium caused

beriberi. Eijkman believed that the beriberi germ or toxin was in the

polished rice and was neutralized by " something " in the polishings.

 

In 1901, Gerrit Grijns (1865–1944; Fig. 2 ), Eijkman's assistant in

Java, continued the studies. He was probably the first to have a clear

conception of beriberi as a deficiency disease and to attempt to

isolate the protective and curative component from foods. Grijns

proposed that the disease was caused by a nutritional deficiency, or

the lack of a specific natural substance found in certain foods.

 

Casimir Funk (1884–1967; Fig. 3 ), a chemist, regarded the Eijkman

factor in beriberi as a definite organic chemical substance, one of

several whose inclusion in trace amounts in an otherwise adequate diet

was responsible for the cure or prevention of deficiency diseases such

as beriberi, scurvy, rickets, and pellagra. In 1911 Funk isolated a

pyrimidine-related concentrate from rice polishings that was curative

for polyneuritis in pigeons (1)(2). His concentrates were primarily

nicotinic acid—not effective for beriberi but later shown to cure

pellagra—contaminated with the antiberiberi factor (3)(4). His

analyses indicated that the concentrate contained nitrogen in a basic

form and was probably an amine. Since it appeared to be vital to life,

Funk named it " vitamine " (5). Although they were not amines, the name

stuck and has been applied to a whole series of substances found in

foods, independent of their chemical structure.

 

In 1920, Jack Cecil Drummond (1891–1952; Fig. 4 ) suggested that,

since there was no evidence to support Funk's original idea that these

indispensable dietary constituents were amines, the final " e " be

dropped, so that the resulting word vitamin would conform with the

standard scheme of nomenclature, which permits " a neutral substance of

undefined composition " to have a name ending in " in. " Drummond also

recommended that the " somewhat cumbrous nomenclature " then in use

(fat-soluble A, water-soluble B, water-soluble C) be dropped, and the

substances be referred to as vitamins A, B, C, etc., until their true

nature was identified(6).

 

Hopkins and Accessory Food Factors

 

Frederick Gowland Hopkins (1861–1947; Fig. 5 ), the father of British

biochemistry and a major contributor to biochemical thought and to

experimental biochemistry, firmly established the existence of

vitamins. He opposed the vitalist thinking of many of his

contemporaries. For him the nature of protoplasm was not mysterious

but something accessible to the experimental approach. He entered

Guy's Hospital Medical School at the age of 27 and distinguished

himself in chemistry. After qualifying, he worked for several years in

the medical school as a laboratory technician by day and as a clinical

chemist in a privately owned clinical research laboratory in the evenings.

 

As a result of his interest in uric acid, along with his early

training and experience as an analyst, Hopkins developed a new and

superior method for its determination in urine (1892) (7). Although

eventually superseded by colorimetric and other methods, Hopkins's

procedure remained the most accurate and reliable for several decades.

Together with Sidney W. Cole, Hopkins tracked down the glyoxylic acid

impurity in the glacial acetic acid reagent responsible for the

already well-known Adamkiewicz reaction of proteins. Consequently,

they modified the reaction by replacing the acetic acid with a

solution of glyoxylic acid. Their work led to the discovery and

isolation of tryptophan (1901) (8)(9), and to the finding that it was

essential for growth. In 1921, Hopkins isolated and named glutathione,

a tripeptide. He also discovered the enzyme xanthine oxidase.

 

In 1898, Hopkins joined the physiology staff at Cambridge University,

but not until 1911, when he was almost 50 years of age, was he able to

devote the greater part of his time to the development of biochemistry

at the university and to his own research. In 1914, Hopkins became

chairman and the first professor of biochemistry at Cambridge, and the

new department became a magnet for biochemists. After Hopkins

introduced biochemistry into the natural sciences curriculum at

Cambridge in 1935, elementary courses in biochemistry became

widespread in English universities.

 

In 1912, Hopkins published what is perhaps the best known of his

works: " Feeding Experiments Illustrating The Importance of Accessory

Factors In Normal Dietaries " (10). He had been impressed by the

conflicting results in the nutritional studies of other workers.

Reasoning that there was more to an adequate diet than the types of

amino acids in the protein, he concluded that normal food must contain

some unknown component that was lacking in a basic synthetic diet made

up of a mixture of purified protein (casein), carbohydrate (starch),

and fats (lard), with mineral salts and water. For some unexplained

reason, young rats fed on such diets failed to grow and even lost

weight unless they were given small amounts of milk daily. Hopkins

reasoned that milk contained " accessory food factors " that are

required only in trace amounts but are indispensable for normal growth

and maintenance.

 

Apparently there were physiological values in natural food products

not indicated by the ordinary methods of chemical analysis and not

included in total energy values that were absolutely essential to

growth, maintenance, and general well-being. The chemical nature of

these physiological values remained a mystery. Consequently, although

Hopkins's paper, and Funk's review a few months earlier, focused

attention on the " vitamine question, " the very existence of vitamins

continued to be in doubt.

 

Discovery of Vitamins A, D, and C

 

The effect of the small additions of milk observed by Lunin,

Pekelharing, and Hopkins was soon recognized to be due to the action

of more than one essential substance. Independent investigations in

the US provided evidence for another growth factor. In 1913, Lafayette

Benedict Mendel (1872–1935) of the Sheffield Scientific School

(affiliated with Yale University) and Thomas Burr Osborne (1859–1929)

of the Connecticut Agricultural Experiment Station in New Haven

discovered a " fat-soluble " accessory food substance that was clearly

distinct from the " water-soluble " factor revealed in the beriberi

studies. Their finding resulted from the comparison of two diets of

purified components fed to white rats. One diet contained dried whole

milk and the other, dried skim milk. The substitution of butter for

some of the lard in the " skim milk " diet prevented the loss of weight

and eventual death of the rats and demonstrated that butter contains a

trace amount of some fat-soluble organic substance that is essential

in nutrition of this animal.

 

Unluckily for Osborne and Mendel, Elmer Verner McCollum (1879–1967)

and Marguerite Davis at the University of Wisconsin reported a similar

observation with rats fed " the ether extract of egg or of butter " 3

weeks before the Osborne and Mendel paper was received for

publication(11)(12). Both papers appeared in the same volume of the

journal. McCollum and Davis were credited for the discovery of the

first accessory food substance to be recognized as a vitamin, which

they called " fat-soluble A. " Both teams had shown by controlled animal

experiments that certain fats contain a factor essential for

nutrition, whereas others do not (11)(12).

 

" Fat-soluble A " was first believed to be a single vitamin capable of

curing xerophthalmia and rickets. Cod-liver oil was first used as a

therapeutic agent in the 1770s. The beneficial effect of fish liver

oils in the treatment of rickets, osteomalacia, generalized

malnourishment, and certain eye conditions was widely recognized by

the middle of the 19th century, but no satisfactory explanation

accounted for its superiority over other edible fats. In 1922,

McCollum et al.(13) showed that cod-liver oil aerated at the

temperature of boiling water for 12 to 20 h retained its antirachitic

activity in rats, but was ineffective against xerophthalmia. In

addition, these properties were unequally distributed in certain

foods. Apparently, two separate factors were involved. The factor

effective against rickets later was named vitamin D. The Wisconsin

workers found that when cod-liver oil is saponified, the vitamin

remains in the nonsaponifiable fraction; therefore, it is a sterol.

 

Meanwhile, advances were being made in the study of scurvy, probably

the first disease to be definitely associated with a food deficiency.

Scurvy was common in northern Europe and for centuries was the scourge

of sailors on long voyages when fresh food was not available. The

symptoms of scurvy are weakness, anemia, pain in the joints, and

hemorrhages from the mucous membranes of the mouth. The gums are

particularly affected by swelling, redness, and ulceration. In 1753,

James Lind (1716–1794), a British naval surgeon, wrote Treatise on the

Scurvy and reported the effective use of orange and lemon juice in

preventing scurvy in sailors and urged this as a standard part of the

diet. In 1795, the government finally added lemon juice to the ration

of the British sailor.

 

In 1907, two Norwegians, Holst and Frolich, produced a condition in

guinea pigs comparable with human scurvy by feeding them a cereal diet

and eliminating fresh animal and vegetable foods. The addition of the

restricted foods to the diet cured the surviving animals(14).

 

The name " water-soluble C " was initially proposed by Drummond(15) in

1919 for the antiscorbutic factor. Albert Szent-Gyorgyi (1893–1986)

isolated this substance in 1928 during enzyme research and renamed it

ascorbic acid. Szent-Gyorgyi received the Nobel Prize for physiology

or medicine in 1937 for his discoveries with special reference to

vitamin C.

 

What soon followed the work of Hopkins, McCollum and Davis, Osborne

and Mendel, and others was a complete revolution in the science of

nutrition. Largely through Mendel's work, nutrition was transformed

from empiricism to a clearly recognized branch of biochemistry founded

upon scientific principles. The American Institute of Nutrition was

formed in 1933. As for Hopkins, he was knighted in 1925 and in 1929

shared the Nobel Prize in physiology or medicine with Eijkman " for

their discovery of the growth-stimulating vitamins. "

 

Year by year, additional factors were discovered and shown to be

necessary for prevention of one kind of disorder or another in humans

or animals. Several of these substances were also needed as growth

factors by microorganisms. Synthesis in the laboratory provided a

product identical in properties and physiological effect with the

" natural " vitamin, and gave rise to a new growth industry of

" nutritional supplements " —an idea subject to much criticism and

controversy.

 

The claim is often made that healthy individuals eating a

well-balanced diet do not need vitamin supplements. However, the

public, increasingly aware of the benefits of vitamins as heralded by

commercial advertisements and publicity in the news media, purchases

these preparations in single and multiple combinations—often at levels

far surpassing the recommended dietary allowance (RDA) for daily

intake published by the National Academy of Sciences. Excessive use of

some vitamins, which is more common in affluent societies, can cause

vitamin imbalance. Vitamin poisoning may occur, especially involving

overdose of vitamin A.

 

Federal agencies, reference laboratories, and industrial manufacturers

are responsible for analyzing vitamin content of foods. Manufacturers

are required to list on packages the vitamin content of processed

foods, especially for A and C. Milk is fortified with vitamins A and

D, and breads and other wheat products are enriched with the B-complex

vitamins. Vitamin supplements are prescribed for expectant mothers and

often for elderly patients. Multivitamins are included in the total

parenteral nutritional mix for patients unable to consume oral

feedings. Although deficiency of a single vitamin is relatively

uncommon in humans, it can occur as a result of an inborn error of

metabolism or from an unusual restriction in dietary intake. More

frequently, complex deficiencies may result from food fads or as

complications from diseases affecting food absorption, as well as in

nutritionally deficient areas of the world. Deficiencies may also

arise from large losses of blood, from hemodialysis, after

gastrointestinal surgery, as a consequence of the use of certain

drugs, or following certain types of treatment such as radiation or

chemotherapy.

 

Analysis of Vitamins

 

n 1926, Carr and Price (16) introduced the reaction of vitamin A with

antimony trichloride in chloroform, in which the blue color produced

soon reaches a maximum intensity and then rapidly fades or changes to

other colors. Under carefully controlled conditions the blue color

persists long enough to make accurate readings possible.

 

Chemical methods for determining vitamin C are based upon the reducing

properties of the vitamin and include titration procedures with

various oxidizing agents. In 1937, Roe introduced a color reaction

with 2,4-dinitrophenylhydrazine to determine vitamin C. In 1943, Roe

and Kuether (17)(18) further developed the method and applied it to

analyses of blood, plasma, and urine. Vitamins A and C were the only

ones commonly determined in the clinical chemistry laboratory.

However, the infrequent number of requests for these tests make it

convenient to refer them to the reference laboratories where vitamin A

is analyzed by HPLC. Vitamin C continues to be analyzed by

modifications of the method with 2,4-dinitrophenylhydrazine, but

fluorometric and HPLC techniques have also been used.

 

The Reality of Vitamins

 

Paul Karrer (1889–1971), in his Nobel Prize lecture for chemistry in

1937 for his investigations on carotenoids, flavins, and vitamins A

and B2, stated that " scarcely ten years have elapsed since the time

when many research scientists doubted the material specificity of the

vitamins and were of the opinion that a special state of matter ...

was the cause of the peculiar vitamin effects which had been observed "

(19). Similar doubts had been expressed earlier in the discussion

about what the enzymes " really are. " These exchanges were part of the

tug of war between mechanists and chemists that recurs on numerous

occasions. The former see all physiological events as mechanical

processes, whereas the latter explain all vital phenomena in

essentially chemical terms. The controversy originates in the search

for answers that would flow from one simple and universal concept and

ends in the recognition that neither of the opposing ideas alone can

provide the answer.

 

During the 1930s interest in vitamins grew, and chemical methods were

sought to replace the very slow and laborious assays involving

animals. When the determination of vitamin A was readily achieved by

an ultraviolet measurement in the range of 320–330 nm, at least five

photometers were developed specifically for this assay. They used line

emission sources that were not applicable to the majority of

ultraviolet analyses. In 1940 the two most popular spectrophotometers

were made by Cenco and by Coleman. They used an incandescent tungsten

source that barely reached the ultraviolet region. Scientists who

wanted ultraviolet photoelectric instruments had to build their own.

 

In early 1940, Arnold Beckman and his colleagues recognized that the

DC amplifier designed for the pH meter could also be used with

vacuum-type phototubes. The company, whose major products were pH

electrodes and meters, began a spectrophotometer development program

that in 14 months resulted in the model DU Quartz Photoelectric

Spectrophotometer(20)(21).

 

The design of the DU was carefully thought out. A prism monochromator

was selected in preference to a grating to minimize stray light. The

instrument featured variable slits, a hydrogen lamp source for the

ultraviolet, and an incandescent automotive headlight bulb (operated

at reduced voltage for stability) for the visible region. Two

phototubes were used, one for the ultraviolet, the other for the visible.

 

The introduction of the DU in 1941 ensured the end of absorptiometry

by means of the spectrograph with its dependence on the tedious,

inconvenient, and imprecise processing and measurement of photographic

plates. Now for the first time an ultraviolet and visible absorption

spectrum could be obtained with relatively inexpensive instrumentation

and within a reasonable time, even though point-to-point readings were

required. The DU greatly accelerated method research in the visible

and ultraviolet range. The DU met a need and was an immediate success.

It remained unsurpassed in its field for 35 years.

 

The contribution of industrial scientists to the development of

clinical chemistry has been one of the characteristics of American

science and may be traced to Arnold O. Beckman, the founder of Beckman

Instruments (Fullerton, CA). Although the two instruments for which he

is best known, the Model G pH meter and the DU spectrophotometer, were

not designed specifically for clinical chemical applications, they

subsequently led to widespread use in acid-base studies and

photometric measurements of many kinds.

 

References

 

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2. Drummond JC, Funk C. The chemical investigation of the

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3. Ihde AJ. Casimir Funk. In: Gillispie CC, ed. Dictionary of

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