( Originally Published 1956 )
The isolation of vitamin B12 and the demonstration that this substance is the anti-pernicious anemia factor of liver was an outstanding achievement (121). Search for this factor had been continuous since the late 1920's when Minot and Murphy demonstrated the efficacy of liver in the treatment of pernicious anemia and Castle proposed his theory of the pathogenesis of this disease. Castle postulated that an intrinsic factor in gastric juice combined with an extrinsic factor in food to form the anti-pernicious anemia factor of liver. Intrinsic factor was found to be absent from the gastric juice of patients with pernicious anemia. It is now known that vitamin 1312 is not only the antipernicious anemia factor of liver but is also the extrinsic factor of food .
Vitamin B12, like many other members of the B complex, is not a single substance but comprises a group of closely related compounds with similar physiologic activity. Vitamin B12 itself is cyanocobalamin, while vitamin B12a,. b, and d, which are identical, are hydroxycobalamin and vitamin B12c is nitritocobalamin. An interesting facet of the metabolism of vitamin B12 is its dependence for absorption, in the amounts present in the diet, on the intrinsic factor of gastric juice. Accordingly, pernicious anemia, which is due to deficiency of vitamin 13,2, does not develop because of dietary inadequacy but because of an insufficient supply of intrinsic factor. It is also of interest that when very large amounts of vitamin B12 are administered orally, absorption occurs in the absence of intrinsic factor.
In spite of extensive investigation, the chemical structure of intrinsic factor is unknown and the mechanism by which it influences absorption has not been elucidated. A number of modes of action have been suggested (125). An attractive postulate is that absorption is regulated by a mechanism similar to that for the absorption of iron (. Intrinsic factor may have functions other than facilitating absorption of vitamin 1312, It is known that most of the vitamin B12 in blood is bound to the alpha and beta globulin fractions of serum protein; very little is present as the free vitamin. Recent studies suggest that intrinsic factor may facilitate the combination of vitamin B„ with a specific protein moiety of serum . Miller and associates (128) have found that intrinsic factor concentrates from hog intestine stimulate the uptake of vitamin B12 by liver slices or homogenates and also by homogenates of kidney and intestinal mucosa. Other studies in our laboratory indicate that there may be a deficiency of the protein factor in serum which combines with vitamin 1312 in some patients with macrocytic anemia. These observations may lead to a better understanding of the pathologic physiology of pernicious anemia and should assist in the development of new tests useful in diagnosis.
Vitamin B12 is undoubtedly an essential human nutrient but present information is insufficient to estimate dietary requirement. The administration of approximately 1 microgram daily, parenterally, will induce remission in pernicious anemia (4a). Presumably, the normal adult needs to absorb an amount no larger than this but the biological availability of vitamin B12 in the diet remains unknown. Vitamin B,2 is required for growth in animals. Studies of the effect of vitamin B12 on growth in human subjects have been equivocal (113). It seems likely that under certain conditions of diet and nutrition, vitamin B12 may have a growth promoting effect.
The metabolic functions of vitamin B12 are gradually being elucidated . This vitamin appears to be involved in the synthesis of nucleoproteins by way of its participation in the metabolism of purines and pyrimidines. Promotion of normal hematopoiesis may be explained on this basis. The mode of action in the metabolism of nervous tissue has not been clarified. Vitamin B12 may have a role in the metabolism of methyl, hydroxymethyl and sulfhydryl groups.
Vitamin B12 Deficiency
From the clinical standpoint, vitamin B12 deficiency is seen primarily in pernicious anemia. Several other types of macrocytic anemia and some neurologic disorders may also be due to deficiency of this vitamin. Until recently, the diagnosis of pernicious anemia has been dependent entirely on clinical and nonspecific. laboratory findings. Characteristically, the disease occurs in well nourished persons of middle age or beyond who give a history of similar illness in other members of the family. Weakness, lemon-yellow pallor of the skin and signs of cerebral anoxemia or congestive heart failure are often presenting findings. Neurologic abnormalities are common, particularly parasthesias of the fingers and toes and signs of peripheral neuritis or subacute combined degeneration of the spinal cord. Gastrointestinal complaints are frequent and glossitis is observed in most instances.
The peripheral blood shows severe macrocytic anemia, leukopenia with hypersegmentation of the polymorphonuclear cells, and thrombocytopenia. The bone mar-row is hyperplastic and contains large numbers of megaloblasts. Excessive erythrocyte destruction is manfested by an increase in the concentration of bilirubin in serum and high urobilin excretion in the urine. Hydrochloric acid is absent from the gastric juice even after stimulation with histamine. An abnormality of tyrosine metabolism is demonstrated by the excretion of certain phenolic compounds in the urine.
While diagnosis is easy in the classical case of pernicious anemia, difficulties often arise in differentiation from sprue and nutritional macrocytic anemia and even from macrocytic anemia in diseases unrelated to nutritional deficiency. Estimation of the concentration of vita-min B12 in serum is of considerable value in diagnosis and in judging the adequacy of therapy (123b, 30). The concentration of vitamin B12 in serum falls to abnormally low levels in pernicious anemia in relapse, even before there is significant decrease in erythrocytes in the peripheral blood. The level returns to normal with therapy. The exact concentration indicative of pernicious anemia is dependent on the method of assay which is employed. With a microbiological assay using L. Leichmanii as the test organism (131), normal serum contains from 80 to about 500 micromicrograms (uug) per milli-liter. In pernicious anemia, serum concentration is below the normal range and in some instances no vitamin B12 can be detected. Normal levels of serum vitamin B12 assayed with the organism Euglena gracilis range from 350 to 750 micromicrograms per milliliter (132). Low levels of vitamin B12 in blood may be observed in macrocytic anemias other than pernicious anemia that will respond to vitamin B12.
The urinary excretion of vitamin B12 has been studied by numerous investigators (110). Register and Sarett (119) found an average excretion of 31 millimicrograms (mpg) daily in normal subjects on ordinary diets. In other studies, somewhat higher values have been re-ported. Excretion tends to be less in patients with pernicious anemia than in normal persons but large variation among individuals prevents this test from being of diagnostic value. Studies of urinary excretion following par-enteral injection of vitamin B12 indicate, in general, an increase in excretion with increasing dosage (123b). Following oral administration of vitamin B12, there is little increase in urinary excretion unless very large doses (1.0 to 3.0 mg.) are administered. Even in these instances, excretion is less than anticipated by the concentration of vitamin B12 in serum. i.e., at levels comparable to those obtained with parenteral doses, urinary excretion is much lower.
A procedure which has been developed for indirectly detecting the presence or absence of intrinsic factor in the gastric juice is the Schilling test (133) which em-ploys vitamin B12 labeled with an isotope of cobalt, Co 60, Two micrograms of the vitamin (0.5 microcuries radio-activity) is administered orally followed in 2 hours by a 1000 µg parenteral "flushing" dose of non-labeled vitamin. Urine is collected for 24 hours and radioactivity measured. In our experience, normal subjects usually excrete more than 6% of the radioactive vitamin with this technique whereas patients with pernicious anemia excrete less than 2%, the amount often being barely detectable. The test is repeated after two or three days using a similar oral dose of Cos 60 vitamin Biz combined with intrinsic factor. Excretion of vitamin B12 will increase to normal levels in patients with pernicious anemia. In sprue and other macrocytic anemias in which intestinal absorption is defective, excretion remains low even in the presence of intrinsic factor.
Other tests of absorption of vitamin B12 include measurement of radioactivity in the feces after oral administration of Co60 vitamin Biz (134) and comparison of radio-activity over the liver after oral and parenteral administration of the labeled vitamin.
An in vitro test for intrinsic factor activity which may be applied to gastric juice has been developed in our laboratory (127). It is dependent upon the ability of intrinsic factor to increase the amount of vitamin B12 which will combine with serum proteins. A procedure for measuring "serum factor" in blood, i.e., the protein moiety which binds vitamin Biz has also been developed. Patients with pernicious anemia show low intrinsic factor activity in gastric juice and often low "serum factor" with these procedures. In some subjects with so-called nutritional macrocytic anemia both tests are extremely low. Further study with these procedures may assist in elucidating the etiology of some of the macrocytic anemias and be helpful in their differentiation.
Vitamin B12 has been found to be effective therapeutically, in several types of macrocytic anemia other than pernicious anemia, including that which follows gastrectomy, the anemia associated with fish tapeworm infestation, some instances of sprue and nutritional macrocytic anemia and, rarely, in macrocytic anemia of chronic liver disease. Following total gastrectomy, vitamin 1312 deficiency develops on the same basis as in pernicious anemia, that is, absence of intrinsic factor. Vitamin B12 deficiency in sprue and nutritional macrocytic anemia may be due to dietary deficiency, impaired absorption, deficiency of serum factor or some other cause as yet unappreciated. The macrocytic anemia of fish tapeworm infestation appears to be due to utilization of vitamin B12 by the parasite in the intestinal tract. In cirrhosis of the liver, storage of vitamin B12 may be impaired.
Vitamin B12 has been used in the treatment of a number of neurologic diseases unassociated with pernicious anemia. This vitamin has been reported to relieve the pain of nutritional neuropathy and to be beneficial, at times, in the therapy of peripheral neuritis associated with diabetes mellitus. In our experience, vitamin B12 has been efficacious in some instances in treating neuritis that occurred in subjects who had chronic alcoholism and cirrhosis of the liver. Whether a deficiency of vitamin B12 is present in these conditions remains unknown. Some abnormality of utilization rather than dietary inadequacy might explain the findings. Massive par-enteral doses of vitamin 1312, 1000 ,ug daily, have been reported to be followed by cessation of pain in trigeminal neuraliga. In this situation, vitamin B12 may be exerting a pharmacologic rather than a physiologic action.