Clinical Chemistry 43: 680-685, 1997;
(Clinical Chemistry. 1997;43:680-685.)
© 1997 American Association for Clinical Chemistry, Inc.
Vitamine—vitamin. The early years of discovery
Louis Rosenfeld
New York University Medical Center, Department of Pathology, 560 First Ave., New York, NY 10016.
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Abstract
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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
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Introduction
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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).
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Hopkins and Accessory Food Factors
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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.
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Discovery of Vitamins A, D, and C
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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.
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Analysis of Vitamins
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In 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.
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The Reality of Vitamins
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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.
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References
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Abstract
Introduction
