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 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 B₁₂ 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'.

Phosphorus Method