Antibody to Transcobalamin II and B12 Binding Capacity in Patients Treated With Hydroxocobalamin

Thirty-two patients treated for B₁₂ deficiency with one or two initial depot seriesof five i.m. injections of 1 mg hydroxocobalamin on alternate days followed byi.m. injection of 1 mg hydroxocobalaminevery third month for maintenance therapy were examined after more than 2 yrof treatment. Antibody to TC II demonstrable after agar gel electrophoresis wasdetected in 5 of 12 patients given twodepot series 1-3 mo apart, while antibody to TC II detectable only after themore sensitive immunoelectrophoresisoccurred in 2 of 20 patients given oneinitial depot series or two series 6 or 12mo apart. No anti-TC II was observed inserum from untreated patients with pernicious anemia.

Submitted on April 26, 1971 Revised on June 24, 1971 Accepted on July 7, 1971     

Transcobalamin II deficiency with methylmalonic aciduria in three sisters

Transcobalamin II (TC II) is a plasma protein that binds vitamin B₁₂ (cobalamin, Cbl) and facilitates cellular Cbl uptake by receptor-mediated endocytosis. In autosomal recessive TC II deficiency, intracellular Cbl deficiency results in an early onset of megaloblastic anaemia that may be accompanied by neurological abnormalities. Inadequate treatment may lead to neurological abnormalities. We describe three sisters, the daughters of first cousins of Moroccan origin, with TC II deficiency requiring continuous and long-term vitamin B₁₂ treatment. The diagnosis was suspected from the finding of low unsaturated vitamin B₁₂ binding capacity and confirmed by absence of detectable TC II by radioimmunoassay and by inability of cultured fibroblasts to synthesize TC II.     

Turnover of 57Co-labelled Vitamin B12-Transcobalamin II and Autologous 131I-labelled IgG in a Patient with Antibody to Transcobalamin II

In a pernicious anaemia patient with a circulating antibody to transcobalamin II (TC II), the plasma clearance of radioactive vitamin B₁₂ bound to TC II has been shown to be much slower (plasma half-life 8.2 days) than in control subjects (plasma half-life 0.7 days). Thus, the patient's high serum vitamin B₁₂ binding capacity and elevated serum vitamin B₁₂ are the result of decreased catabolism of TC II due to the presence of antibody. The turnover of the B₁₂-TC II-antibody complex was shorter than that observed for autologous ¹³¹I-labelled IgG (plasma half-life 16.8 days). This difference is most likely due to dissociation of the complex.     

Hydroxocobalamin. I. Blood Levels and Urinary Excretion of Vitamin B12 in Man After a Single Parenteral Dose of Aqueous Hydroxocobalamin, Aqueous Cyanocobalamin and Cyanocobalamin Zinc-Tannate Complex

A single intramuscular injection of 500 or 1000 µg. of hydroxocobalamin to17 individuals resulted in a 1.8- to 4.1-times higher mean serum vitamin B₁₂blood level, respectively, 5 hours after injection; a 4.6- and 12.8-times higherlevel 24 hours after injection; a 2.4- and 5.2-times higher level 72 hours afterinjection, and a 1.6- and 2.4-times higher level by the 2nd through the 4thweek after injection than identical doses of cyanocobalamin administered to19 individuals. The vitamin B₁₂ blood levels following i.m. administration of500 or 1000 µg. of hydroxocobalamin were significantly higher during the first24 and 48 hours, respectively, than they were after a cyanocobalamin zinc-tannate complex given to 17 individuals at identical doses.

After a single i.m. injection of 500 or 1000 µg. of hydroxocobalamin, anaverage of only 16 per cent and 27 per cent, respectively, of the vitamin B₁₂was lost in the 72-hour urines, as compared to 60 per cent and 69 per cent,respectively, after identical doses of cyanocobalamin. These differences, again,were highly significant statistically.

The results of these studies give evidence of a slower rate of urinary excretion of hydroxocobalamin as compared to that of cyanocobalamin, and ofits ability to build up consistently higher and more prolonged vitamin B₁₂levels in the blood.

Submitted on June 6, 1961 Accepted on August 1, 1961     

Methylmalonic Acid Excretion in Vitamin B12 Deficiency

methylmalonic acid was first isolated from pooled human urine in 1957 by Thomas and Stalder and convincing evidence has since been obtained to show that in man the isomerization of l-methylmalonyl-Co A to succinyl-Co A is dependent on the B₁₂ coenzyme dimethylbenzimidazolyl cobamide (White, 1962). Indeed, Cannata, Focesi, Mazumder, Warner and Ochoa (1965) showed recently that l-methylmalonyl-Co A mutase bears two molecules of cobamide coenzyme firmly attached to its molecule. In a series of 30 patients Cox and White (1962) detected excessive methylmalonic acid in the urine of all patients with serum vitamin B₁₂ levels below 140 pg./ml., their lower limit of normal. Patients with normal serum B₁₂ levels excreted up to 4 mg. of methylmalonic acid in 24 hours. Their series included six patients who were treated with intramuscular hydroxocobalamin (1000 μg.). In five of these, excretion fell to normal within 3 days, and on the fifth day after injection in the sixth patient. Barness, Young, Mellman, Khan and Williams (1963) reported one vitamin B₁₂ deficient patient in whom the urinary excretion of methylmalonic acid reached 500 mg. in 24 hours. Recently Khan, Williams, Barness, Young, Shafer, Vivacqua and Beaupre (1965) have described seven out of nine patients with pernicious anaemia in whom methylmalonic acid excretion persisted for some months after the institution of B₁₂ therapy. This occurred despite full clinical and haematological remissions and in four patients, normal serum vitamin B₁₂ levels. The present study was undertaken to determine the value of urinary methylmalonic acid excretion as a screening test for vitamin B₁₂ deficiency. The estimation of methylmalonic acid was carried out by a rapid thin layer chromatographic technique, suitable for routine laboratory use. Normal controls and patients suffering from general haematological and medical disorders were screened as well as patients suffering from megaloblastic anaemia. In several patients the excretion of methylmalonic acid was followed for a short period after B₁₂ therapy.     

Forms of Vitamin B12 in Blood and Bone Marrow in Patients with Pernicious Anaemia

The biologically-active forms of vitamin B₁₂ in blood and bone marrow and changes induced in these by injections of cyanocobalamin have been measured in patients with pernicious anaemia. Bone marrow methylcobalamin was low before therapy, and increased 24 h after therapy. The largest portion of bone marrow vitamin B₁₂ was 5′deoxyadenosylcobalamin, and this increased more than did methylcobalamin during the 24 h after injection of cyanocobalamin. A single injection of roo μ of cyanocobalamin induced about 10 times the increase of the intracellular coenzyme forms of vitamin B₁₂ in bone marrow than followed injection of 100 μg. Plasma methylcobalamin was extremely low before therapy, and increased only moderately 24 h after therapy; the majority of plasma vitamin B₁₂ remaining as cyanocobalamin. In contrast, only a minority of intracellular bone marrow vitamin B₁₂ was cyanocobalamin 24 h after injection of cyanocobalamin. The degree of anaemia did not correlate with bone marrow methylcobalamin, nor did bone marrow cobalamin correlate significantly with cobalamin content of washed blood erythrocytes. Correlation was observed between intra-erythrocyte vitamin B₁₂ content and the degree of anaemia; the correlation being inverse with haemoglobin concentration in the peripheral blood. Inverse correlation also was observed between MCV and erythrocyte folate content. These studies suggest that megaloblastic maturation appears at different concentrations of bone marrow vitamin B₁₂ in different patients, presumably because in vitamin B₁₂ deficiency, eventual limitation of normoblastic maturation may be determined by factors such as folate metabolism, vitamin B₁₂ binders, and the affinity of vitamin B₁₂ dependent enzymes. Although the clinical response to 1000 μg of cyanocobalamin does not differ from that to 100 μg, the concentration of vitamin B₁₂ coenzymes in bone marrow cells was proportional to the cyanocobalamin injected.     

Assessment of Body Stores of Vitamin B12 During Maintenance Therapy of Pernicious Anaemia with Depot Cyanocobalamin Preparation (Betolvex®)

The serum vitamin B₁₂ level during the first eight weeks after a single injection of the depot-cyanocobalamin preparation, Betolvex, may be considered as a reflection of the body stores. Experiments with this method seem to indicate that patients on maintenance therapy with Betolvex have obtained a replenishment of the tissue stores, while this does not seem to be the case in some patients examined on maintenance therapy with hog's stomach, liver extract, aqueous cyanocobalamin and hydroxocobalamin.     

Antibody to Transcobalamin II in Patients Treated with Long Acting Vitamin B12 Preparations

⁵⁷Co-labelled vitamin B₁₂ bound to transcobalamin I, transcobalamin II or non-protein bound, has been mixed with serum from patients with pernicious anaemia treated with long acting vitmain B₁₂ preparations. By paper electrophoresis, immunoelectrophoresis and Sephadex G-200 gel filtration, an antibody of the γG-globulin type reacting with transcobalamin II was demonstrated. By immunoelectrophoresis the antibody was demonstrated in 8 of 19 patients. Gel filtration experiments showed that the affinity of the antibody to TC II is very sensitive to changes in pH, ionic strength and temperature.     

Inheritance of transcobalamin II (TC II) in two families with TC II deficiency and related immunodeficiency

Transcobalamin II (TC II) is an essential transport protein for vitamin B₁₂ in blood. TC II can be separated into isoproteins by polyacrylamide gel electrophoresis. This method was used in combination with a specific radioimmunosorbent technique to evaluate genetic variants and inheritance of TC II-deficient genes in relatives of two children with congenital TC II deficiency. Both patients presented with impairment of haematopoietic and immunological functions. Seven heterozygous individuals for TC II deficiency, who are clinically normal, were detected in the two families. Two out of seven could be identified unambiguously by TC II isoprotein analysis, as carriers of a deficient gene, which does not express functional TC II. Application of this new method to detect heterozygous carriers of the deficient gene provides a valuable addition to genetic counselling.     

Orthostatic Hypotension as a Manifestation of Vitamin B12 Deficiency

A 90-year-old woman with orthostatic hypotension and near-syncope was found to have a low-normal level of vitamin B₁₂ and no other medical findings that could explain her orthostasis. Her symptoms responded to vitamin B₁₂ replacement therapy. This case shows that vitamin B₁₂ deficiency can induce orthostatic hypotension and syncope that are correctable by vitamin B₁₂ replacement.Key words: Hypotension, orthostatic/etiology/therapy; vitamin B 12/therapeutic use; vitamin B 12 deficiency; vitamin B 12 replacementOrthostatic hypotension (OH) is a common disorder, especially among elderly persons. Patients with OH usually undergo neurologic, cardiologic, and endocrine evaluation, because there are several causes for this presentation. One of these causes, vitamin B₁₂ deficiency, is well known among neurologists but is often overlooked by cardiologists. We describe a case of OH in an elderly woman whose condition improved with vitamin B₁₂ replacement therapy. This case brought the attention of our cardiology group to this vitamin deficiency and its importance as a potential diagnosis in cardiology patients.     

Elevated serum homocysteine as a predictor for vitamin B12 or folate deficiency

Abstract: Tissue deficiency of vitamin B₁₂ and folate results in an increase in serum homocysteine (sHcy). We have measured sHcy in patients with reduced serum vitamin B₁₂ and/or red cell folate (RCF) to determine its usefulness as a discriminant for the diagnostic interpretation of reduced vitamin levels. Of 3846 patients who had serum vitamin B₁₂ and RCF assayed, 335 (9%) had reduced vitamin levels. Multivariate analysis showed a significant association between sHcy and serum creatinine (p = 0.0001), positive intrinsic factor (IF) antibody or neutrophil hypersegmentation (NHS) (p = 0.001), increased MCV (p = 0.014) and low RCF (p = 0.025) but no relationship with the level of serum vitamin B₁₂ or haemoglobin. After censoring the patients with renal impairment (n = 54), the distribution of the remaining 72 patients with elevated sHcy was 37/151 (25%) with low serum vitamin B₁₂ with or without low RCF and 35/130 (27%) with low RCF alone. sHcy correctly identified response to vitamin therapy in 33/35 (94%) patients who had adequate parameters to assess response. The positive predictive values of IF antibody/NHS, macrocytosis and/or low RCF for elevated sHcy were 100% and 34% respectively. Twenty-four percent of patients with a low serum vitamin B₁₂ and elevated sHcy had no abnormal haematologic parameters as determined by the routine laboratory staff. These data suggest that the usefulness of measuring sHcy in a routine diagnostic setting is limited and a careful review of the peripheral blood for macrocytosis and NHS plus determination of RCF may be a more cost-effective process than sHcy assay in most instances to determine the presence of tissue deficiency.     

In vivo plasma binding capacity for cyanocobalamin

Many investigators have alluded to or described the presence of multiple vitamin B₁₂ binding proteins in human plasma (Mendelsohn, Watkin, Horbett and Fahey, 1958; Miller, 1958; Hall and Finkler, 1962; Rosenthal, Hill and Haessler, 1964; Heller, Epstein, Cunningham, Henderson and Yakulis, 1964). Recently, Hall and Finkler (1963, 1964) separated two cyanocobalamin plasma binders with distinctly different characteristics. One fraction (Transcobalamin II or TC II) is apparently a β-globulin which is involved in the immediate carrier state, while the other (Transcobalamin I or TC I), an α-globulin, is the endogenous B₁₂ binding protein. Gabuzda, Worm-Petersen and Lous (1965) confirmed these findings, demonstrating that cyanocobalamin is bound in vitro by a distinctly separate protein from that to which it is bound endogenously. Methods derived from a study of plasma binding by vitamin B₁₂ analogues have been utilized to determine in vivo plasma binding capacity for cyanocobalamin (Meyer, Schiffer, White and Cronkite, 1965). It is proposed that this in vivo plasma binding capacity represents to a large extent the B₁₂ carrying capacity of TC II.     

Screening for vitamin B12 deficiency in psychiatric patients

Psychiatric patients are frequently screened for vitamin B₁₂ deficiency in the absence of hematologic or other neurologic findings. To determine the yield of this practice, 162 psychiatric inpatients were screened for vitamin B₁₂ deficiency. Ten patients had initial low serum vitamin B₁₂ levels, but only two had definite B₁₂ deficiency on further evaluation. Three patients who had initially low B₁₂ levels had normal levels subsequently during outpatient follow-up. When low serum vitamin B₁₂ levels are discovered in psychiatric patients without hematologic or neurologic findings, a diagnosis of B₁₂ deficiency should not be presumed without further evaluation. Received from the Department of Medicine, New England Deaconess Hospital, and Harvard Medical School, Boston, Massachusetts.     

Measuring holotranscobalamin II, an early indicator of negative vitamin B12 balance, by radioimmunoassay in patients with ischemic cerebrovascular disease

Circulating homocysteine is a risk factor of cardiovascular and cerebrovascular events. Hyperhomocysteinemia may be an early indicator for vitamin B₁₂ disorders because cobalamin is a cofactor in the remethylation process of homocysteine. Serum holotranscobalamin (holoTC II) becomes decreased before the development of metabolic dysfunction. In this study, we assessed circulating holoTC II to estimate the diagnosis of vitamin B₁₂ deficiency in the first ischemic cerebrovascular attack. We also compared the efficacy of the measurement of plasma holoTC II with the other standard biochemical and hematological markers used to reach the diagnosis of cobalamin deficiency. Forty-five patients (age 71 years (range 35–90), 16 men/29 women) within the first ischemic cerebrovascular event were included in this prospective study. All the enrolled patients have been administered vitamin B₁₂ 1 mg intramuscular injection once a day for 10 days. At the baseline and on the tenth day of treatment, plasma levels of holoTC II and the proper biochemical and hematological markers in diagnosing cobalamin deficiency were measured. After admission, anemia and diminished serum vitamin B₁₂ levels were determined to be only 20% (9/45) and 44% (20/45), respectively; 78% (35/45) of the patients had low serum holoTC II (<37 pmol/l). Serum homocysteine was higher in patients (49% of them) who had previously suffered a stroke. Thrombocytopenia, hypersegmentated neutrophils, and indirect hyperbilirubinemia were observed in 20% of the patients. Leukopenia and macrocytosis were not evident in any of them. In 18 of 27 patients (67%) that had low holoTC II levels after joining the study and who remained in the study until the end of cobalamin treatment, serum holoTC II levels returned to normal values. Cobalamin deficiency should be considered in patients with cerebrovascular diseases, even if anemia, elevated mean cell volume, depression of the serum cobalamin, or other classic hematological and/or biochemical abnormalities are lacking. Furthermore, measurement of serum holoTC II looks promising as a first-line of tests for diagnosing early vitamin B₁₂ deficiency.     

Hydroxocobalamin. II. Absorption from the Site of Injection and Uptake by the Liver and Calf Muscle in Man

Labeled hydroxocobalamin, administered intramuscularly in man, is absorbed more slowly from the site of injection and deposited in the liver andcalf muscle to a degree equal to or higher than that of cyanocobalamin at anequivalent dose. These findings, together with those reported in Part I ofthis paper, suggest the applicability of hydroxocobalamin as a long-actingvitamin B₁₂ in man. These conclusions, however, require the support to be obtained from long-range observations on patients with vitamin B₁₂ depletion.

Submitted on June 6, 1961 Accepted on August 1, 1961     

Malabsorption and deficiency of vitamin B12 in HIV-infected patients with chronic diarrhea

Deficiency of vitamin B₁₂ is commonly reported in HIV-infected patients. We measured vitamin B₁₂ levels in 36 HIV-infected patients with chronic diarrhea (>3 stools/day for six weeks or more). Eight patients had an identifiable cause of diarrhea. Vitamin B₁₂ levels were low in 39%. Sixteen of these patients were selected to undergo further testing, eight patients with low levels of vitamin B₁₂ and eight with normal B₁₂ levels. These 16 patients had both a stage II Schilling test and measurement of multiple serumd-xylose concentrations performed after both oral and intravenous doses ofd-xylose. Integrated areas under the curves (AUC) ford-xylose concentration versus time were calculated for intravenous and oral doses, andd-xylose bioavailability was determined. Stage II Schilling tests were abnormal in 11 patients, (69%).d-Xylose bioavailability correlated closely with vitamin B₁₂ absorption (r=0.648,P<0.01). Comparisons of mean values for CD4 count, serum albumin, Karnovsky score, six-month weight loss, 1-hr serumd-xylose levels and MCV failed to reveal a significant difference between those with and without abnormal serum vitamin B₁₂ levels. These data indicate that below-normal levels of vitamin B₁₂ are highly prevalent in HIV-infected patients with chronic diarrhea. Malabsorption of vitamin B₁₂ occurs in the setting of an enteropathic process effecting both the proximal and distal small bowel. Since no risk factors for vitamin B₁₂ deficiency could be identified, screening for vitamin B₁₂ deficiency in HIV-infected patients with chronic diarrhea is strongly recommended.Supported in part by grant RR00048 National Institutes of Health, National Center for Research Resources.     

Reversible Dementia and Neuropathy Associated with Folate Deficiency 16 Years After Partial Gastrectomy

A 57-year-old woman developed dementia and peripheral neuropathy 16 years after a partial gastrectomy (Billroth II). Serum cobalamin was 198 pmol/l (reference interval 150–550), and the vitamin B₁₂ absorption test (Schilling) showed decreased absorption (1.7% without and 2.2% with intrinsic factor). In spite of 20 months' therapy with vitamin B₁₂, the neurological symptoms progressed. Folate deficiency was suggested by a very low erythrocyte folate and a slightly abnormal FIGLU test. There were no other signs of general malabsorption. A few months' treatment with folic acid significantly improved the massive neurological manifestations which were verified neurophysiologically as well as histologically. A common role of vitamin B₁₂ and folate in the development of neuropathy is suggested.     

The lymphocyte as a marker of past nutritional status: persistence of abnormal lymphocyte deoxyuridine (dU) suppression test and chromosomes in patients with past deficiency of folate and vitamin B12

In short-term suspension cultures of bone marrow cells or PHA-stimulated lymphocytes from normal subjects, non-radioactive deoxyuridine (dU) suppresses the incorporation of radioactive thymidine ([³H]TdR) or its analogue, [¹²⁵I]deoxyuridine ([¹²⁵I]Udr), into DNA. This normal suppression by deoxyuridine (dU) is impaired in both of these cell systems from patients with deficiency of folate or vitamin B₁₂, and corrected by the appropriate vitamin. Patients with megaloblastic anaemia due to deficiency of vitamin B₁₂ or folate were studied before and after treatment. When treatment had returned to normal the bone marrow morphology and the serum and red cell vitamin levels, then the dU suppression test and chromosomal changes in the bone marrow were also corrected. However, the dU suppression test and chromosomal changes remained abnormal in lymphocytes as long as 84 d after therapy. These abnormal lymphocyte dU suppression tests were corrected by the appropriate in vitro additions of folic acid, methylfolate and vitamin B₁₂, depending on the vitamin deficiency present before therapy. These studies suggest that an abnormal lymphocyte dU suppression test corrected by the appropriate vitamin in vitro, and characteristic chromosome abnormalities in lymphocytes, when these are absent in the bone marrow, indicate past deficiency of vitamin B₁₂ or folate. These changes can be used for retrospective diagnosis of these deficiencies in patients treated by ‘shotgun’ therapy. They further support the concepts that circulating unstimulated lymphocytes: (1) do not incorporate appreciable amounts of vitamin B₁₂ or folic acid; (2) reflect the vitamin status of the patient at the time the lymphocytes were generated; and (3) cannot replace bone marrow in dU suppression tests aimed at diagnosis of current marrow and other non-lymphocyte cell line nutrient status. These studies add to the evidence that selective nutrient deficiency may occur in one but not another cell line in the same person, and point to the need for more studies on factors affecting nutrient delivery, uptake, and utilization by various human cell lines. These studies also provide a new approach to evaluation of circulating lymphocyte age.     

Incidence of gastric parietal cell antibody in families of patients with iron deficiency anaemia

There is an association of pernicious anaemia with iron deficiency anaemia (Faber and Gram, 1924). There is also a high incidence (85 per cent) of gastric parietal cell antibody in the sera of patients with pernicious anaemia (Taylor, Roitt, Doniach, Couchman and Shapland, 1962) and a less marked, but significantly increased incidence (33 per cent) in patients with iron deficiency anaemia who have a histamine fast achlorhydria (Dagg, Goldberg, Anderson, Beck and Gray, 1964). Pernicious anaemia has a familial incidence (Askey, 1940; Callender and Denborough, 1957) and there is also an increased frequency of achlorhydria in relatives of patients with pernicious anaemia (Askey, 1940; Neel, 1947). In addition te Velde, Abels, Anders, Arends, Hoedemaeker and Nieweg (1964) have found the gastric parietal cell antibody in 20 per cent of relatives of patients with pernicious anaemia, compared with 6 per cent in a control series. Because of the association of iron deficiency anaemia with pernicious anaemia and the increased incidence of histamine fast achlorhydria and parietal cell antibody in both diseases, the occurrence of the antibody has been assessed in the families of patients with iron deficiency anaemia, histamine fast achlorhydria and gastric parietal cell antibody. In a group of 22 patients with iron deficiency anaemia, histamine fast achlorhydria and gastric parietal cell antibody, Dagg, Goldberg, Gibbs and Anderson (1966) found that seven had a ⁵⁸Co vitamin B₁₂ absorption test (Schilling, 1953) characteristic of pernicious anaemia, and six of these had reduced serum vitamin B₁₂ levels; that is these patients had latent pernicious anaemia. In 10 of the 22 patients the serum vitamin B₁₂ levels and the Schilling test were normal, and in the remaining five patients the serum vitamin B₁₂ was low but the Schilling test was normal. Since it appeared possible that the familial incidence of gastric parietal cell antibody would be higher in relatives of the patients with latent pernicious anaemia than in relatives of those without latent pernicious anaemia, 11 families from these 22 patients were investigated, six of which had propositi with latent pernicious anaemia and five of which had propositi without latent pernicious anaemia (Table I). The relatives of the remaining 11 patients were unwilling to co-operate in these investigations.     

Crude or Refined Intrinsic Factor in Preparations for the Oral Treatment of Pernicious Anaemia

A report is given of a comparative study of the absorption of vitamin B₁₂ with the aid of crude or purified intrinsic factor preparations. A crude hog's pylorus material, contained in Hepaforte®, was shown to have an intrinsic factor activity, in relation to its ₁₂ binding capacity, about 40 per cent of that in a purified material used in Bendogen®. No qualitative difference in intrinsic factor activity could be traced for the two materials studied. In pernicious anaemia patients, refractory to standard treatment with vitamin B₁₂ + intrinsic factor, the B₁₂ absorption is generally in the normal range, when the amount of intrinsic factor material given is sufficiently high. Long term clinical trials show, however, that some refractory patients show subnormal serum B₁₂ values even after intrinsic factor doses much higher than those generally recommended. Also in these studies no fundamental difference was found between crude and purified intrinsic factor materials. Oral treatment of pernicious anaemia with vitamin B₁₂ plus intrinsic factor has no place in modern therapy, especially in view of our present knowledge that a daily oral dose of 1 mg vitamin B₁₂ without intrinsic factor gives full protection in all cases of B₁₂ deficiency.