A history of pernicious anaemia

This is a review of the ideas and observations that have led to our current understanding of pernicious anaemia (PA). PA is a megaloblastic anaemia (MA) due to atrophy of the mucosa of the body of the stomach which, in turn, is brought about by autoimmune factors. This is not a review of MA in general, nor of other forms of cobalamin (Cbl) deficiency that can also lead to a MA. Following the recommendation of a nomenclature commission, the name Cbl is used rather than vitamin B12. Progress comes with the acquisition of new facts either by new ideas, new methods, new measuring devices, availability of new science, etc. Once new tools appear several groups will apply them to their common problems and credit often has to be shared. With apologies to James Combe of Edinburgh (1796–1883), Thomas Addison of London (1793–1860), Antoine Biemer of Zurich (1827–1892) and others, it is not possible to make a clinical diagnosis of PA among the case reports appearing in the first three-quarters of the nineteenth century. No mention of a smooth or painful tongue, not a touch of icterus, not a needle or pinprick sensation, let alone numbness, among the lot, make it difficult to accept a diagnosis of PA. Nevertheless, physicians were writing about a disease that often was PA and making admirable observations and perceptive suggestions about its causation. A case report by Osler & Gardner (1877) in Montreal could be that of PA. This anaemic patient had numbness of the fingers, hands and forearms; the red blood cells were large; at autopsy the gastric mucosa appeared atrophic and the marrow had large numbers of erythroblasts with finely granular nuclei. The increased marrow cellularity had also been noted by Cohnheim (1876). The key that was missing was a method of scrutinizing the blood. This arrived through the efforts of two cousins, Carl Weigert and Paul Ehrlich. Weigert developed staining of tissue sections with the new aniline dyes, while Ehrlich, still a medical student, applied these methods to staining heat-dried blood films placed on a copper plate, one end of which was warmed by a bunsen burner. Ehrlich (1854–1915) had been given a corner in the hospital basement by Wilhelm Waldeyer (1837–1921) and when he asked Ehrlich what he was doing was told ‘Iche probe’, which may be translated loosely as ‘I am messing about’. Among other observations, Ehrlich (1880)(Fig 1) distinguished between cells he termed megaloblasts present in the blood in PA from normoblasts present in anaemia as a result of blood loss. Paul Ehrlich (Wellcome Institute Library, London). Not only were large red blood cells noted in PA, but irregular red cells, ? poikilocytes, were reported in wet blood preparations by Quincke (1877). Megaloblasts in the marrow during life were first noted by Zadek (1921). Hypersegmented neutrophils in peripheral blood in PA were described by Naegeli (1923) and came to be widely recognized after Cooke's study (Cooke, 1927). The giant metamyelocytes in the marrow were described by Tempka & Braun (1932). The association between PA and spinal cord lesions was described by Lichtheim (1887) and a full account was published by Russell et al (1900), who coined the term ‘subacute combined degeneration of the spinal cord’ (SCDC) although they were not convinced of its relation to PA. Arthur Hurst at Guy's Hospital, London, confirmed the association of the neuropathy with PA and added, too, the association of loss of hydrochloric acid in the gastric juice (Hurst & Bell, 1922). Cabot (1908) found that numbness and tingling of the extremities were present in almost all of his 1200 patients and 10% had ataxia. William Hunter (1901) noted the prevalence of a sore tongue in PA, which was present in 40% of Cabot's series. Many had wondered whether PA was the result of some inadequacy of diet; food supplements were tried with limited success, but some forays gave a flicker of hope which, alas, was not confirmed by others. George Hoyt Whipple (1878–1976) studied the rate of haemoglobin regeneration in dogs made anaemic by venesection; thereafter different foods were compared to test their efficacy in restoring the haemoglobin level. Liver was the most effective. George Richard Minot (1885–1950), like Whipple, became interested in the role of diet in relation to anaemia, particularly PA. He spent much time taking dietary histories and was impressed by the frequency with which patients limited themselves to a restricted diet that excluded meat. He advised patients to include meat and liver in the diet and, in some patients, improvement appeared to follow. Minot (Fig 2) had been shown a method of counting reticulocytes by James Homer Wright (1869–1938) that was useful for assessing blood responses and he persuaded a colleague, William Murphy, to join him. In 1923, they started a regimen that included 100–240 g of liver and 120 g of ‘muscle meat’, leafy vegetables, fruit, eggs and milk taken daily. Some patients noted clinical improvement and their reticulocytes increased within 4–5 d followed by rises in red cell count and haemoglobin levels. It took them 2 years to treat 45 patients who could tolerate the diet and all these responded (Minot & Murphy, 1926). George Richard Minot (Wellcome Institute Library, London). In 1934, the Nobel Prize in medicine and physiology was awarded to Whipple, Minot and Murphy. Was there ever an award more deserved? They saved the lives of their patients and pointed the way forward for further research. What was there in liver that was lacking in patients with PA? The effect of liver in restoring the anaemia in Whipple's iron-deficient dogs was by supplying iron which is abundant in liver. Liver given by mouth also provides Cbl and folic acid. But patients with PA cannot absorb Cbl, although some 1% of an oral dose can cross the intestinal mucosa by passive diffusion; this, presumably, is what happened when large amounts of liver were eaten. Beef liver contains about 110 μg of Cbl per 100 g and about 140 μg of folate per 100 g. Cbl is stable and generally resistant to heat; folate is labile unless preserved with reducing agents. The daily requirement of Cbl by man is l-2 μg. The liver diet, if consumed, had enough of these haematinics to provide a response in most MAs. Soon, liver concentrates were developed that could be given by mouth (Fig 3) and, subsequently, by injection, and rescued patients from a liver diet that, indeed, was horribly unpalatable. However, sensitivity to the animal protein present in the liver extract given by injection was the next problem and was only overcome when its use was replaced by the pure antianaemic factor. A prescription which includes extract of half a pound of liver daily which was renewed for 2 years. This patient stopped treatment for pernicious anaemia in 1940 and was found to have severe MA again in 1961. The availability of liver extracts brought about interest in the nature of the haematological response. An optimal response required a peak rise of reticulocytes 5–7 d after the injection of liver extract and the height of the peak was greatest in those with severe anaemia; the flood of reticulocytes was as a result of a synchronous maturation of a vast number of megaloblasts into red cells. There is a steady rise in the red cell count to reach 3 × 1012/l in the 3rd week (Minot & Castle, 1935). Many liver extracts did not have enough antianaemic factor to achieve this and some assayed by the author had only 1–2 μg of Cbl. It took another 22 years for a pure antianaemic factor to be isolated, although, admittedly, the Second World War intervened; in 1948, an American group led by Karl Folkers and an English group led by E. Lester-Smith published, within weeks of each other, the isolation of a red crystalline substance termed vitamin B12 and subsequently renamed cobalamin (Lester-Smith, l948; Rickes et al, 1948). The slow progress was because the fractions that were isolated from liver had to be tested for activity in untreated PA patients, although this step was bypassed in the final stages using a microbiological assay with Lactobacillus lactis for the antianaemic factor by the American group (Shorb, 1947). The structure of this red crystalline compound was studied by the nature of its degradation products and by X-ray crystallography. It soon became apparent that there was a cobalt atom at the heart of the structure and this heavy atom was of great aid to the crystallographers, so much so that, with additional information from the chemists, they were the first to come up with the complete structure. To quote Dorothy Hodgkin: ‘To be able to write down a chemical structure very largely from purely crystallographic evidence on the arrangement of atoms in space – and the chemical structure of a quite formidably large molecule at that – is for any crystallographer, something of a dream-like situation’. As Lester-Smith (1965) pointed out, it also required some 10 million calculations. In 1964, Dorothy Hodgkin was awarded the Nobel Prize for chemistry. However, this is not the whole story. Unknown to the scientists who had worked on the isolation of Cbl, the use of cyanide to produce cyanoCbl, as well as exposure to light, had knocked off an important structure. Barker et al (1958) published an account of the metabolism of glutamate by a Clostridium. The glutamate underwent an isomerization and an orange-coloured co-enzyme was involved that turned out to be Cbl with a deoxyadenosyl group attached to the cobalt. This Cbl co-enzyme, deoxyadenosylCbl, is the major form of Cbl in tissues; it is also extremely sensitive to light, being changed rapidly to hydroxoCbl. DeoxyadenosylCbl is concerned with the metabolism of methylmalonic acid in man (Flavin & Ochoa, 1957). The other functional form of Cbl is methylCbl involved in conversion of homocysteine to methionine (Sakami & Welch, 1950). Both these pathways are impaired in PA in relapse. Cbl consists of a ring of four pyrrole units very similar to that present in haem. These, however, have the cobalt atom in the centre instead of iron and the ring is called the The have a further a termed at to the and this may have a to the cobalt atom the time Cbl had been isolated from liver it was that it was also present in as gave so that of pure Cbl were these have replaced liver in the of Cbl. a form of cobalt to the instead of Cbl became et al, 1950). The of Cbl is that it made it possible to out Cbl in patients, to for Cbl, to of factor to to and to as Cbl is by the any by the It also forms the for to Cbl. The most useful is Cbl with which provides the of but has a with a peak that provides a of counting the nineteenth it was that there was acid present in the gastric juice and that it was not for this to be in PA patients Hurst of acid in the gastric juice in PA and that it the of anaemia by years. & found hydrochloric acid in the gastric juice from of PA the of gastric juice in PA is much and not increased by a to The association of and PA was confirmed by methods for gastric and of making a diagnosis of PA. The of acid in the gastric juice a diagnosis of PA. of the gastric mucosa in PA in was reported by at the London He also found impaired of by an extract of the gastric mucosa in PA, in with an extract of a & into the or stomach after and the first of gastric atrophy the of the but the The in the study of gastric and intestinal was in by the use of a by et al in In PA, the mucosa is replaced by cells and there is and cell It be that the stomach are not to PA and are present in who are and not PA also have atrophy is by of the & which is by in the gastric and It is the major of gastric The of acid into the gastric in is the for of The present in PA, as well as in severe gastric atrophy PA, in loss of the and, very in of William at the Hospital, to the between gastric the antianaemic factor that was also present in and the response in PA. The asked was it possible that the stomach of the could something from food that for was to The in untreated patients with PA of two of 10 d or more during which daily were the first of 10 the PA patient g of each There was response. the the of the stomach of a man were after the of g of about 100 g could not be The gastric were for a and given to the PA patient through a This was daily. there was a rise in reticulocytes a peak on followed by a rise in the red cell The response was similar to that with large amounts of oral liver. that of gastric juice given alone to untreated PA patients each did not produce any response. gastric juice was not that the factor in gastric juice was labile et al, that a was taking between an factor in the gastric juice and an factor in Minot & found that g of liver daily was to a response in PA, 10 g liver was when with gastric juice & factor is the as the antianaemic factor that is Cbl, and is for its the gastric juice in PA & as well as & that is more resistant to and when it is combined with Cbl and this has been confirmed by The of et al in using of sections of stomach with that was in the gastric The of Cbl to the cell was by first the with a to The cell in man is the of hydrochloric acid and The cell is the only of in man as a is followed by a MA due to Cbl is a with a of 45 An assay for was described by & and et al juice has two that can to Cbl. The one is and the other is the present in all body and which to have The of gastric juice can be by the of Cbl it but to has to be is the Cbl in gastric juice in PA. of Cbl by gastric juice is is to a further of gastric the the on Cbl can only to the of gastric juice 100 of Cbl. the of an it only of Cbl, which is a of the of of Cbl is as a result of to A of was as the that took up of Cbl. the had an of units of per gastric In these an of Cbl was and Cbl was the method being to as used by & The of in gastric juice in man is about in the and to an of 10 units or after a of gastric juice as etc. The is about units or units are required for of a μg dose of Cbl in PA. The in is about of that in Following a to gastric the of a peak in the first as compared with hydrochloric acid which at 45 The peak is because of a of With of a to the of level. were by In PA there is a in and of The of in PA 3 units per of gastric juice compared with units in and the in PA an is to a of to of of μg of Cbl & As loss of from the gastric juice to be the of PA, it to treat patients by them oral were from stomach and to have all but out of PA patients on an oral for years. preparations of were and of about 100 of with 10 μg of Cbl were However, & in London and et al in the noted that the of Cbl in these patients after about a followed by of a In time it was shown that the was as a result of the of the is a found in and it has an requirement for Cbl. It was used by et al for the assay of the antianaemic factor and the assay was by for the assay of Cbl in a of assay have been used with and, methods have replaced of the microbiological found that the had to be in to Cbl from in it could be taken up by that was a in at Hospital, London, and he with in the & that all untreated patients with PA had an Cbl level. This has one of the major for the from the With of patients with of all patients with Cbl deficiency have a Cbl. However, life is Cbl in the of any in the blood or A Cbl although present in untreated PA, not there are other for a Cbl than PA of protein fractions of after that Cbl is in the of of after of on that Cbl was attached to two one the termed and the other after the termed that, when Cbl given by mouth is it first in the of and in the of as well and They that is the Cbl protein Cbl from the into the blood and to the liver from it is by new as well as of a functional a severe MA in the first of life to an to Cbl. of the Cbl in is on because it has a of the of is about in the Cbl it is that is being In untreated PA, the very Cbl on the to more Cbl than is with Cbl. too, is one of the With the availability of Cbl, Cbl to be widely used in the The method was the test described by an oral dose of Cbl is followed by an injection of μg of The is largely into the the next and with it about one of the Cbl. With a μg dose of Cbl, 10% or more of the oral dose is in the by But there are other more of assessing the whole body counting the μg injection of Cbl is and the in a taken about 10 after the oral these methods not on the patient a complete In PA, there is impaired of Cbl and it is when the test is with Cbl given with oral The of the test deficiency as the of the Cbl The improvement of Cbl in PA when the test is with generally not reach the found in and may be so as to the of intestinal as the of the Cbl This is to the of at but it be that can also result in a & noted that the result of a test with a μg dose of Cbl in was In patients with PA, the result with was of these that, in the was from and gastric the were similar to with a of Among another PA patients, was present only in the was In 10 PA patients with present only in gastric the with was and, in PA patients was found in and gastric the test result with was the more abundant the the is the response to There is a further the of Cbl with in PA may be The of Cbl in man is the et al, & 1957). In untreated PA, it is years the for the have as much as a molecule of and it can several for of the to be This of has been termed Cbl and in a of patients with Cbl not only in PA but also in Cbl deficiency in etc. The is also the of the that Cbl into blood et al, There is a frequency of PA among those that have the among patients with also had PA as compared with a frequency of PA of about per in the patients with also had PA. The association is among the out of patients with also had PA. with PA and pure red cell have been In the in the first or of PA, and disease themselves and all cell et al, are present in in of different of and in the of of the of PA The is present in from of patients with of patients with of of patients and of patients with cell are found in between of the being in There is a frequency of PA in the to being to The cell is important in the of gastric are present in from of in from of PA in of from patients, in of in disease and in of of patients with on patients had to oral treatment with preparations that their when given by mouth with to Cbl in a The activity was in the of the an oral to of Cbl with 10 of from PA patients who had not been to also the of Cbl by these This was the case with from out of patients with PA The in the PA had the in observations were translated into an in test by & They that the of Cbl to in gastric juice was by PA that had to This was the case with out of a of PA tested in 10 published et al found in out of gastric juice from PA and from in gastric juice and in out of gastric juice from PA patients who did not have a had an in the gastric in gastric juice that were tested were and et al that an had the end of an intestinal are almost this to the stomach as the of the intestinal et al that there was an of cells in the gastric mucosa in PA. with are to make have PA as This cannot be as a result of of and be because of & in the of in out of PA patients and & James found 10 out of were was by & James with a test for factor of blood cells from the end of a is in the of This test was in out of PA patients were In of patients with PA had a result for four patients with and PA tested had to patients with PA were tested for the of in and gastric juice and for but one gave in one or more It was that these the autoimmune nature of PA and that the is not an are often in and the to a in one out of PA patients is and with PA with a of the the they are still the anaemia will in the first et al, but in the term Cbl neuropathy may be The of Cbl and may & There is a of in the gastric juice and and a in the of in In some patients there is of acid in the gastric a of and cells & this is as a result of of the cell and of to a slow to the The author has into the two by the the of a & and and

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