Folic Acid came out of
Yellapragada SubbaRow's search of 18 long years for APAF
the anti-pernicious anaemia factor.It is really the
antidote for tropical sprue which had taken him to the
jaws of death and carried away brother Purushottam-not for
pernicious anaemia. Its failure to tackle the neurological
abnormality in pernicious anaemia caused him to resume the
APAF search in liver. He succeeded but lost publication
priority to a rival team which isolated what is known as
Vitamin B12 from a microbial source. Folic Acid and B12
supplement each other in the treatment of a wide range of
megaloblastic anaemias including pernicious anaemia and
tropical sprue.
Folic
Acid remains fascinating as it is of fundamental
importance to cellular metabolism: it provides the
methyl group in the synthesis of methionine so essential
to life processes and it has an important role in the
formation of precursors to DNA and RNA which hold the
keys to heredity.
After
graduating from the Harvard School of Tropical Medicine,
SubbaRow settled down in the biochemistry department of
Harvard Medical School to unravelling the role of
phosphorus in living beings.
SubbaRow's
expertise in removing protein from liver before
extracting phosphorus compounds interested fellow
Harvardian Gordon Alles as the two got acquainted one
September 1927 evening in the dining room of the
Vanderbilt Hall. Gordon was an assistant to Edwin
Cohn the authority on protein chemistry.
George
Minot and William Murphy, the Boston physicians who had
won the Nobel for their miraculous cure for pernicious
anaemia, had approached Cohn to make the medicine,
broiled pig liver, palatable to their patients.
The doctors had difficulty force feeding their
appetite-lacking patients with a hundred grams daily of
the evil-looking meal. They wanted Cohn to extract
from liver the substance that cured pernicious anaemia.
SubbaRow
and Alles met regularly comparing notes on their
differing methods of removing protein from liver.
Soon the initial interest in each other's techniques led
to each acquiring interest in the other's
objective. Alles moved into insulin research in
which SubbaRow had previously worked and eventually left
for California where years later he died of all things
undiagnosed diabetes. And SubbaRow moved into
Alles' field isolating the pernicious anaemia cure from
liver. For doctors those days believed that both
pernicious anaemia and tropical sprue had a common cure
because they were so alike in symptoms and signs, and it
was opportunity to avenge the death of Purushottam.
Bernard
Jacobson, a medical student helping SubbaRow in
phosphorus research, would get their liver supply from
butchers glad to get rid of it at a special price.
And SubbaRow would heat and boil liver with chemicals
and came up with differing coloured and smelling
solutions. Jacobson would then feed them to guinea pigs,
drew blood, smeared glass slides and counted red blood
corpuscles under a microscope. In 1930 Cohn
dropped his APAF work after producing an injectable
liver extract for patients. Jacobson was by then
settled in Massachusetts General Hospital with a
research fellowship and began to administer PA patients
in his hospital SubbaRow's liver extracts which boosted
red blood corpuscles in guinea pigs. And, when a 3
m.g fraction brewed by SubbaRow from hundred grams
of fresh liver radically improved the blood picture of
their animals, the duo was excited thinking they had
reached the end of their long labours. The liver
concentrate so potent in animals was worthless in
patients! SubbaRow abandoned the animal test and
had the patients given the solution from which he had
precipitated the concentrate. The patients
benefited.
SubbaRow
used charcoal to decolourise the solution before
harvesting crystalline material from it. The
crystals were only a third as active as the
solution. Instead of blaming charcoal for loss of
activity, SubbaRow reasoned that charcoal had perhaps
soaked up all that was good in liver for Jacobson's
patients. If his hunch was right, he had indeed
made a breakthrough. To prove it, he had first to
extract what had got bound (adsorbed) on to the surface
of the charcoal. It took weeks to test each of the
possible extractors with the equipment he himself
fabricated.
In
April 1934, Vilma Prochownick, a German refugee student,
walked into his life and work. She began to
question his leisurely pace of charcoal extraction with
crude equipment. She suggested vacuum pumps and
was not put off when he countered: 'What is the hurry
with distillation? It gives me time to think and
read.' She took it up with Cyrus Fiske, SubbaRow's
professor, and was told the depression-strapped Harvard
could not increase the departmental budget. Her
enthusiasm toned up SubbaRow and his work gathered
speed. By September he recovered with common ethyl
alcohol the liver goodness stolen by charcoal. The
recovered liver fraction was consistently beneficial to
Jacobson's patients. SubbaRow's hunch had been
right on mark.
Amidst
all this excitement that winter came a visitor to the
Department. Guy W Clark had been helped the year
earlier by Nobel Laureate William Murphy to put out for
Lederle Laboratories a liver injection for pernicious
anaemia patients. The 'Lederle 3 C.C. Parenteral'
was approved by the American Medical Association and was
received well by the medical profession but sediment was
soon found in the vials. Clark rushed to Murphy
and was referred to Fiske. He cautiously asked
Fiske if he could be of any help to SubbaRow in his
efforts to 'unravel liver'.
'Why
don't you,' Fiske said remembering Vilma's
importunities. "Here he is cooking up to 30
to 40 pounds of liver under great difficulty while you
have all sorts of equipment at Lederle.'
Under
an informal arrangement, Clark began to get technical
advice from SubbaRow in return for a steady supply of
crude liver extracts and vacuum pumps Vilma desire for
the distillation equipment.
And
in May 1935, Jacobson was able to present to the Society
for Clinical Investigation data on a little brown 1 cc
water solution of three crystalline liver fractions
derived from, and as effective as, 100 gms of the
original Minot and Murphy prescription of raw
liver. A month later Clark brought on market the
'Lederle 1 C.C. Liver Extract'. Vilma was
delighted that SubbaRow's work, which she had assisted
and helped speed up, yielded a preparation that was
getting into general medical practice. But she was
now stricken with a pulmonary infection and had to leave
SubbaRow and abandon research altogether.
Life
for SubbaRow centred again on the laboratory. He
went Friday evenings to Pearl River and directed the
next two days Guy Clark's men to process half a ton of
liver in the Lederle plant normally idle over the
weekend. He worked hard the week days in Boston on
fractionating the liver concentrates he took to Boston
in a couple of gallon jugs. He achieved nothing
more than a little jump now and then in the liver
extract potency in spite of all the energy and time he
expended, all the devotion with which the men in Pearl
River sacrificed their weekends and all the facilities
and funds the big corporation provided. Fraction I which
brought about blood regeneration with 0.7 mg daily doses
in Jacobson's patients was the ultimate in the
concentration of APAF with the analytical methods then
available.
SubbaRow
now applied his analytical mind to the problem of
isolating APAF. Five of the chemically distinct
compounds he isolated from liver were inactive by
themselves but brought about in patients a response
approximating that of crude liver when administered
along with Fraction I. SubbaRow and Jacobson in a
1937 paper called Fraction I the primary factor and the
five the accessory factors. A year later SubbaRow
advanced a 'multiple factor hypothesis' casting doubt
whether pernicious anaemia was caused by the lack, and
would hence be cured by the administration, of a single
substance and raising the possibility that the effective
cure of pernicious anaemia rested on the interaction of
several factors. Jacobson presented their
'multiple factor therapy to the 1940 New York session of
the American Medical Association. They could get
their rivals at the session agree with them only that
the hypothesis could be proved by the isolation of APAF
in its pure crystalline form alone. None of the
doctors had any idea that APAF was present in liver not
in milligrams but in micrograms. Fraction I had
cut the liver dose by 1/100,000; less than a milligram
of it was as effective as the original 100 grams of raw
liver. But there are a thousand micrograms to a
milligram! It would be another eight years before
new micro techniques would finally zero in on the
elusive APAF.
SubbaRow's
move at this time to Lederle Laboratories was
opportunity no doubt to pursue the pernicious anaemia
factor with greater vigour and resources. But he
as research director could not confine himself to this
quest. And, to his greater glory, he didn't.
But he wasn't done with liver yet. At least not
done with the residue left after pernicious anaemia
concentrates were extracted at Lederle for what was
still a profitable line.
For
the original idea that both pernicious anaemia and
tropical sprue were variants of the same nutritional
deficiency and could be cured by the same vitamin had
long since been challenged by a study among expectant
mothers in Bombay. There Dr Lucy Wills and Dr
Manek M Mehta of the Haffkine Institute found that
the anaemic poor women had a sound digestive system and
that tropical sprue manifested among them when pregnancy
or illness increased the demand for the unidentified
food factor their blood needed. This was in
contrast to Boston's pernicious anaemia patients whose
degenerate stomach walls could not absorb the
blood-building factor present in their rich food.
When Dr Wills later reported that sprue in monkeys was
cured not by APAF-rich liver concentrates but by the
cruder liver extracts, SubbaRow had even in 1937 turned
his attention to the liver plant wastes.
Then
a sprue patient of Dr Jacobson got better with an
APAF-rich liver extract. Instead of casting doubts
on Lucy Wills's claims, SubbaRow decided to test every
liver fraction in both pernicious anaemia and tropical
sprue patients. Since sprue is not common in the
United States, he set up the sprue investigation
programme in Puerto Rico with Dr Ramon M Suarez in
Hospital Mimiya at Santurce.
SubbaRow's
liver men at Pearl River were put to work on the first
liver extract Suarez reported beneficial. They
kept concentrating the 'sprue factor' along the lines
encouraged by reports flowing in from Santurce
only to find at the end of two years that their material
contained mostly of the already identified vitamins of
the B Complex. The 'sprue factor' had been lost
somewhere while being concentrated.
As
luck would have it, a liver fraction SubbaRow was also
testing as a chicken growth promotion factor turned out
at this time to be the true sprue factor. SubbaRow
brought over to Pearl River in October 1941 the man who
had originally traced the 'chick growth factor' in
yeast. With the arrival of Dr E L R Stokstad from
Petaluma in California, SubbaRow got the last element
for success. He could buy unlimited quantities of
liver and yeast, and his pilot plants could process a
thousand pounds of them at a time. And the
concentration of the factor could be followed with two
microbes which also thrived on it. And he himself
with his unrivalled knowledge of fractionation
techniques and organic procedures could guide Stokstad
who was eager to succeed and young enough not to have
any fixed notions about the path to success.
SubbaRow
started Stokstad on a tar-like waste from the liver
plant. Each milligram of it had 20 units of the
chick growth factor. Stokstad's task was merely to
concentrate the active material just 50,000 fold.
Stokstad
easily removed a mass of inert material from liver tar
and ran the extract up a glass column filled with
activated clay and the active material separated out in
a distinct colour zone on the column. On extraction with
alcoholic ammonia, it was found to have 340 units of the
growth factor per milligram--a 17-fold
concentration. This was the new technique of
chromatography, an improvement on charcoal adsorption
SubbaRow had used in his Harvard work on APAF.
SubbaRow's
appreciation of organic chemistry techniques now came
into play. If the factor, by now called folic
acid, could be converted into its ester or alcoholic
salt, the oily substance would separate out while the
impurities remain dissolved in water. Since direct
esterification of folic acid was not possible, they
converted it into its barium salt. The ester then
obtained had a potency of 3450 units. In three
further steps, Stokstad raised the potency from 3450 to
370,000 to 660,000 to 1,030,000 units. He
converted the methyl ester back to free Folic Acid on
April 5, 1943.
It
had taken Stokstad 21 months and Lederle had spent
100,000 dollars on the effort. But the Folic Acid
yield from liver was however too low for it to be of any
practical value. A thousand pounds of pork liver
costing 255 dollars gave 60 mg of Folic acid, hardly the
size of a pea and no more than three days' requirement
of a normal adult.
SubbaRow
decided to scrap the project. He had found a
richer source of Folic Acid.
Late
one afternoon a year earlier in June 1942 SubbaRow was
walking down to the laboratory with Henry Piersma
carrying a desk-drawer full of flasks with cultures of a
riboflavin-producing microbe. It was either
SubbaRow or Piersma who pointed out one of the flasks
and exclaimed: 'Isn't it amazing how much riboflavin
this organism produces?' It was just fantastic.
The other said, 'I wonder if this organism produces any
other vitamin. The first replied, 'Perhaps I
should take a sample of this culture medium and let Al
Dornbush run an assay.'
SubbaRow
was waiting the next morning at the laboratory and told
Piersma, 'Imagine, that sample is full of folic
acid.' A contaminant in that particular culture
was obviously producing folic acid. Luck was again
favouring the prepared mind. But hard luck!
The contaminant could not be isolated as Piersma had
sterilised the flask after taking out the sample for
Dornbush.
SubbaRow
asked Dr Brian Hutchings, a Wisconsin biochemist he had
hired because of his previous work on folic acid, to
capture the contaminant from bottles and tanks in
Building 62 where riboflavin was being fermented.
Hutchings
exposed Petri plates smeared with yeast extract and
glucose in the building and incubated them for 24 to 488
hours. Of the several bacterial colonies that
developed one was a medium-length rod. And,
presto! this benign cousin of the diphtheria germ
produced folic acid.
Hutchings
developed a simple medium in which the bacterium grew
quickly and from which folic acid could be easily
harvested. Fermentation experts grew it in 200
gallon tanks and fermented large batches of broth.
Hutchings used Stokstad's methods to isolate folic acid
from the broth but crystallisation defied him. SubbaRow
put on the job Nestor Bohonos, a biochemist taken off
another project, and he got the crystals within days.
Microbial
yields were big compared to liver yields but SubbaRow,
even as he ordered scaling up the job to manufacture,
decided not to depend entirely on fermentation
broths. He decided in August 1943 to use
crystalline folic acid from Hutchings's laboratory to
attempt synthesis of the vitamin. It was a wise
decision. A year later the pilot plant was still
beset with problems the technicians could not
tackle. SubbaRow took the technical problems upon
himself, put 20 people on investigating fermentation in
all its phases, and provided pilot plant people with
data to get over the difficulties. They were soon
producing 15 grams of folic acid a week at an average
cost of $200 a gram. Large tanks using
cheaper ingredients for fermentation were set up with
hopes of bringing down costs to four dollars a gram but
new snags arose. Although these too were overcome,
SubbaRow wound up the operation on April 28, 1945.
His organic chemists had synthesised folic acid!
But
it had been touch and go. Shortly after SubbaRow
initiated synthetic studies in August 1943, Gustaf
Carlson, his chief organic chemist, left Pearl
River. Neither getting a replacement nor accepting
a suggestion to bring in Vincent du Vigneaud of biotin
fame as consultant, SubbaRow decided to directly
supervise folic synthesis. Initially he did not
even deploy organic chemists. Biochemists Stokstad
and Hutchings broke the folic molecule and found two of
its constituents: glutamic acid from which gluten the
wheat protein is built up and a fluorescent pigment akin
to xanthopterin the yellow colour of butterfly
wings. When the pigment, the 'pteridine' nucleus
of the molecule, defied identification, SubbaRow brought
in the organic chemists, John Mowat and Jim
Boothe. The mystery only deepened when they broke
the folic molecule different ways and got two different
pteridines and speculated about the presence of two
pteridine nuclei in the molecule. They however
found in the non-pteridine part of the molecule not only
glutamic acid but para-amino-benzoic acid (PABG) a
member of Vitamin B Complex, and figured out the linkage
of the two in the folic molecule. When they too
could get no clue as to the identity of the pteridine,
the synthesis itself providing a clear picture of the
vitamin molecule began to be considered.
PABG
and Triamine (TA) used in the synthesis of xanthopterin
were obviously the chemicals to start with. The
chemical had to be chosen that would react with TA to
complete the pteridine nucleus, take on PABG at the
right place and provide the speculated 'fourth carbon
fragment'.
On
Monday the third of July 1944, Mowat brought to the lab
his weekend notebook and got it authenticated for
several ideas including di-bromo-propion-aldehyde as the
third chemical. Later in the day he entrusted the
job of preparing the aldehyde and attempting synthesis
to Coy W Waller who had just been hired by SubbaRow from
the University of Minnesota.
Waller
prepared the aldehyde, reacted it with TA, condensed the
product with PABG and got a brown mud. He asked
for and got the permission to try it all over again
substituting the aldehyde with mucobromic acid which too
had two bromium atoms but with an extra carbon that
could provide the mysterious fourth carbon of the folic
acid. When he did it the customary stepwise
fashion, the mucobromic and PABG formed a glutamate tat
would not readily go into reaction with TA. To
force the reaction, he put the mixture on a Friday
evening in a 'sealed bomb', deciding to keep it cooking
for 72 hours.
Waller
went Sunday afternoon with some friends including
Beverly Braun, who would later become his wife, on
bicycle riding. During dinner at end of the
picnic, he felt anxious about the Bomb and announced he
would go back to the lab to make sure the reaction
wouldn't go too far. Miss Braun went with him,
saying she would start some new cultures for next day's
job in the bacteriology lab where she worked. Whom
did they come face to face at Waller's lab but SubbaRow
who was there also to see everything went well.
The straight-laced director called Waller away and
Beverly Braun, standing anxiously in the lab, could hear
him speak crossly.
The
'sealed bomb' was no success but by changing the
reaction steps, Waller got 0.1% folic activity.
His hopes roused, he asked himself, could it be that
during the mucobromic-PABG reaction a compound was
formed that would, if it got no time to decompose, react
with TA and form folic acid. He went to SubbaRow
and asked if he could throw all the three chemicals
together and allow them to cook in the same soup.
The procedure would be unorthodox but SubbaRow took a
look at the persevering chemist and said, 'Coy, try
anything. Just get it.'
Waller
cooked all the three together in a kettle and got 'a
black, crude and gawky stuff'. He was too
fastidious to send that for assay. It might have
remained on the shelf like the brown mud had not
SubbaRow walked in and said, 'Why not run a bioassay on
this stuff?' The analysts said the stuff had 'one
percent activity--may be two per cent or so'.
But
mucobromic acid was not a satisfactory reagent and a
four-carbon compound like it was not needed as carbon
dioxide escaped during the reaction proving there was
after all no fourth carbon in folic acid. It
occurred to Waller to resurrect the brown mud lying on
the shelf since July and have it assayed. It had
0.75% activity! Waller now got hold of the
original three-carbon Aldehyde proposed by Mowat and
threw it along with PABG and TA in his 'shot-gun'
reaction. What he now got had 15% activity and a
subsequent batch showed 20% activity.
Mowat
phoned the good news to Bound Brook where the chemists
of Calco, sister division of Cyanamid, were following
the work at Pearl River so they could take up process
development and production the moment research was
completed.
Major
chemical activity shifted to Bound Brook. Within a
week, the Calco chemists prepared the first 'laboratory'
batch of 1.7 grams of 15% pure folic acid. The
first pilot plat batch of 4763 grams of folic acid was
ready on July 6. This was 997 grams of 'real'
folic acid. The entire Pearl River group drove
over in SubbaRow's car to get 'a kilo of folic acid all
in one bottle'. Regular manufacture began on
September 26, the first batch yielding 14 kilograms of
'real' folic acid. Twelve kilos were shipped to
Pearl River on December 15.
The
chemical synthesized in Spring was on the manufacturing
line in Fall. A gram of synthetic folic acid cost
$8.70 compared to $123 for fermentation folic acid and
$4250 for liver folic acid.
A
joyous Bell was impatient to announce the synthesis in
March itself but was restrained by patent attorneys to
wait until July 18. That day he sent a telegram to
the annual conference of the American Association for
the Advancement of Science (AAAS) offering research
workers limited supplies for investigating its vitamin
properties.
When
they were permitted to disclose minimum scientific data
in professional journals, Sub's 'Boys' discussed the
paper among themselves and decided to list all the
sixteen who had contributed, at Pearl River and Bound
Brook, to the success in the alphabetical order of their
names. The Boys thought SubbaRow had been no mere
supervisor to have his name put in the last but was one
whose contribution had been 'as much as, perhaps more
than anyone else's'. In the Pearl River listing,
SubbaRow's name appeared just above Waller's when the
paper was published in the August 31, 1945 issue of Science
the journal of three As and one S. SubbaRow
took little interest in all this. He was
preoccupied with the question, 'What value is folic
acid?'
There
were any number of volunteers to provide the
answer. Among them were Tom Spies the old fighter
against 'starvation sickness' as he called the
nutritional deficiency disorders and William J Darby of
Vanderbilt University School of Medicine at Nashville,
Tennessee.
Spies
was amazed when five pernicious anaemia patients showed
an upsurge of well being within four days and
practically a normal blood picture by the eighth day.
Darby
reported the effectiveness of folic acid against sprue,
the old enemy of SubbaRow: A 51-year-old patient with a
ten-month history of sore mouth, diarrhoea, weakness and
weight loss responded spectacularly. Mouth sores
disappeared on the fourth day, the blood picture was
normal on the ninth day and the appetite was
excessive. The man was in excellent health
when he came three months later for a check up.
Equally impressive was the response of other patients.
Spies
now took folic acid to Puerto Rico for extensive trial
in sprue among the patients of the Ramon Suarezes,
father and son. He went on to Havana, Cuba and set
up a trial at General Calixto Garcia Hospital. He
gave folic acid in all to 218 anaemic patients in the
course of a year and hailed it as the fourth element in
his mixed vitamin therapy for starvation sickness.
Despite
the confirmation by other doctors of the initial finding
of Spies in pernicious anaemia, SubbaRow was far from
convinced that folic acid was the long-sought
APAF. The highly purified anti-pernicious anaemia
fractions of liver showed no chemical resemblance to
folic acid. SubbaRow on the other hand had no
grounds to discourage physicians who were resorting more
and more to folic acid in pernicious anaemia since
patients develop sensitivity after a while to liver
injections. This led to a totally unexpected
consequence. An increasing number of relapses and
severe progress of nerve disorders among the patients
soon came to be reported. The New England
Journal of Medicine issued in November 1947 an
editorial warning against the use of folic acid not only
in pernicious anaemia but in sprue.
Careful
studies however established that folic acid, which is
present in the blood and tissues of all normal
individuals, has no bad effect on the nervous
system. Folic acid cures the blood disorder in
pernicious anaemia but has no effect on the associated
nervous disorder which runs its course.
It
not only cures sprue but has caused no nerve damage
among the countless millions it has cured of sprue and
other forms of anaemia. With folic acid, SubbaRow
really avenged the death of Purushottam.
Recently, FDA
approved folic acid for expectant mothers and in fact
recommended its regular use a month before planning
pregnancy to stave off neural tube defects in the
progeny.
***
Folic
acid trials confirmed that pernicious anaemia with the
nerve changes it brings about is quite distinct from
sprue and other megaloblastic anaemias.
Although
he had neglected it after moving to Pearl River,
SubbaRow had not altogether abandoned his search for
APAF in liver. He always had someone working on
liver specifically to try out his ideas on APAF.
He did not know it but he had in fact obtained what was
probably his richest APAF fraction within months of
shifting to Lederle Laboratories. Set by him to
employ the best of classic liver fractionation
techniques, Frank Ablondi had got a beautiful pink
solution. Pink is also the colour of Reinecke
Salt, a toxic chromium compound, used to precipitate the
inactive material. SubbaRow was astonished the
colour remained despite his having broken down Reinecke
Salt with silver nitrate after it had served its
purpose. Believing that all traces of the
poisonous salt had not been removed, he had the solution
extracted with methyl alcohol. Along with the
colour most f the activity got washed out.
SubbaRow
tightened procedures to ensure removal of Reinecke Salt
but Ablondi still got a pink solution. He shook
his head and said reproachfully, 'Frank, you didn't get
the Reinecke out.'
Nestor
Bohonos replaced Ablondi but try as he did he just could
not get active fractions which were not pink. Otto
Weiland replaced Bohonos on the sole task of obtaining
an active liver fraction that wouldn't be pink.
Weiland cried desperately after a year, 'Why use
Reinecke salt at all if it is so risky?' SubbaRow
abandoned it in favour of metallic sulphates.
Weiland ran the resulting solution up the
chromatograph. The pink fraction on the column
boosted blood formation in chicks. Not trusting
the animal test and unable to get over his colour
fixation, SubbaRow did not allow the pink substance to
be given to pernicious anaemia patients.
After
it was finally shown that folic acid was not APAF,
SubbaRow was willing toward the end of 1947 to face the
fact that pink and APAF activity went together. He
was ready to lift the ban on its clinical testing.
The
opportunity to test came on Christmas Eve when Spies
cabled from Cuba that he had a pernicious anaemia
patient. Not finding Weiland anywhere, SubbaRow
waited at his house until six in the evening when the
isolationist returned happily, his arms full of
Christmas gifts. The festivities at home spoiled,
Weiland went to the laboratory and rushed to La Guardia
airfield in New York City to fly out the pink fraction
to Spies. The 65-year-old Cuban patient, admitted
in hospital in a confused state of mind, recovered
fast. Weiland forgave SubbaRow on learning that 30
drops of his Christmas Eve preparation gave back the
Cuban his appetite on the second day, enabled him to
walk on the 25th day and made him soon well enough to go
home.
When
on April 2, 1948 came a similar glowing report about one
c.c. f the same 'special liver fraction' administered to
a woman patient in Alabama, Weiland was glad his
troubles with SubbaRow --working nights, weekends and
holidays--would soon end.
What
ended was SubbaRow's long search for APAF on April
16. That day's issue of Science carried a
Merck Research Laboratories' claim that it had isolated
from liver a crystalline compound which in micrograms
induced positive blood response in pernicious anaemia
patients. It later turned out that Merck got this
Vitamin B
12 not from liver -- although it was
technically possible to get it so as an expensive
curiosity -- but from fermentation broths of the
antibiotic grisein aided by a microbiological assay
acquired quite fortuitously.
Although
Vitamin B12 is without doubt the APAF, it was
folic acid in liver that cured the patients of George
Minot and William Murphy and set in motion the search for
the pernicious anaemia cure. Those unhappy patients
with degenerate stomachs could not have assimilated the
0.06 mg of B12 in the half pound of raw beef
liver they were daily fed.
SubbaRow
in his success in getting out that original Minot-Murphy
talisman and showing it was not the real pernicious
anaemia cure had roused fresh interest in APAF not only
in himself but in other vitamin hunters. His
pioneering fractionation work and his finding that
fermentation broths are good vitamin sources inspired
those who placed 'the last stone and stepped across the
terra firma of accomplished discovery'. |
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