Serum Lysozyme and Vitamin B12 Binding Capacity in Myeloproliferative Disorders

Summary Estimations of serum lysozyme (muramidase), total B₁₂ binding capacity (TB₁₂BC) and folate were performed in 122 patients: 69 with myeloproliferative disorders (39, polycythaemia rubra vera (PRV); 10, chronic granulocytic leukaemia, Ph¹-chromosome positive (CGL); 20, myelosclerosis) and 53 with other disease which affect leucocyte production or turnover (17, monocytic leukaemias; 20, megaloblastic anaemia; 15, neutropenia; and a single case of red cell aplasia progressing to a myeloproliferative disorder). The highest serum lysozyme levels were found in monocytic leukaemia. Serum lysozyme concentrations were also elevated in myelosclerosis and to a lesser extent in CGL and in PRV. High levels in PRV occurred in patients developing myelosclerosis or with active disease, resistant to treatment. Significantly higher concentrations of lysozyme were present in patients with myelosclerosis following PRV than in patients with the primary form of the disease. Patients with myeloproliferative diseases and high lysozyme concentrations tended to have low serum folate levels. Slightly raised levels were found in about 50% of patients with megaloblastic anaemia. Moderately high serum lysozyme concentrations also occurred in patients with neutropenia and hypercellular bone marrows but not in those with hypocellular marrows. TB₁₂BC concentrationswere increased in the myeloproliferative disorders, especially in CGL and to a lesser extent in myelosclerosis and least of all in PRV. The results suggest that serum lysozyme reflects granulocyte turnover (except in monocytic leukaemia) while TB₁₂BC (providing liver function is normal) correlates closely with the total blood (and possibly marrow and splenic) granulocyte pool. The ratio between serum lysozyme and TB₁₂BC proved to be a useful index of the balance between granulocyte turnover and total granulocyte pool in individual patients.     

Vitamin B12 Absorption by Inhalation

Cyanocobalamin in the form of an aerosol was inhaled by normal subjects, patients with pulmonary diffusion defects and patients with vitamin B₁₂ deficiency. In all groups there was a rapid increase in serum B₁₂ levels suggesting that absorption had occurred by diffusion through the lung alveoli.
Six patients with pernicious anaemia given daily inhalations showed complete clinical and haematological remission and a group of six patients with B ₂ deficiency were maintained in remission for 12 months. While effective, this method of administration of vitamin B₁₂ is not regarded as superior to that of injections and is therefore considered to have no therapeutic application. The possibility of inducing pulmonary damage is considered.     

Absorption and Transport of Vitamin B12

A review of recent advances is presented, mostly dealing with physiological mechanisms and the chemical and functional properties of the transport proteins. A new nomenclature is proposed for the R-proteins and transcobalamin II. Scepticism is expressed as to whether we are warranted in accepting the different R-proteins as separate entities.     

Solubilized Receptor for Vitamin B12—Intrinsic Factor Complex from Human Intestine

S ummary . A specific receptor for intrinsic factor-vitamin B₁₂ complex has been prepared in soluble form from human intestinal mucosal cell membranes employing Triton X-100. The extraction procedure has not detectably altered its characteristics relative to membrane-bound receptor. It appears to be macromolecule containing critical peptide and disulphide bonds.     

Megaloblastic Anaemia of Infancy and Vitamin B12

The haematological status of 30 infants with a syndrome characterized by satisfactory growth, abnormal pigmentation, developmental retardation, hypotonia of muscles, hepatosplenomegaly, with or without tremors, has been studied. Anaemia of a moderate degree was seen in all. All but four had megaloblastic erythropoiesis. Serum vitamin B₁₂ levels were below 100 pg./ml. By increasing the infants' intake of vitamin B_12>, by raising the concentration of the vitamin in maternal milk, megaloblastic erythropoiesis could be corrected to normoblastic erythropoiesis. The other clinical manifestations of the syndrome also responded to the administration of vitamin B₁₂ to varying degrees.     

Binding of Vitamin B12-Rat Transcobalamin II and Free Vitamin B12 to Plasma Membranes Isolated from Rat Liver

When dialysed rat serum which contains a single, low molecular weight binder for vitamin B12, rat transcobalamin II (rat TC-II), was labelled in vitro with 57Co-vitamin B12 and then incubated at 30 degrees C (pH 7-5) with vesicles of highly purified plasma membranes separated from microsomal fractions of rat liver by density gradient centrifugation, the 57Co-vitamin B12-rat TC-II complex bound to high affinity sites on the vesicles via a specific (binding after correction for 'non-specific' binding in the presence of a large excess of the non-radioactive complex), saturable, and reversible interaction. The apparent affinity constant for the binding reaction was 5-5 X 10(9) M-1. Using the same incubation conditions, free vitamin B12 also bound to the vesicles of plasma membranes via a specific, saturable, but apparently irreversible interaction. Preincubation of the membranes with free vitamin B12 did not interfere with the subsequent binding of the vitamin B12-rat TC-II complex to the membranes; however, preincubation with the vitamin B12-rat TC-II complex did interfere, to some extent, with the subsequent binding of free vitamin B12. Dialysed rat serum, perhaps the free rat TC-II in the dialysed serum, also inhibited the binding of the vitamin B12-rat TC-II complex to the plasma membranes. The relationship of the binding sites identified in this report to the absorption of vitamin B12 by rat liver, and thus their physiological significance remains unknown until further work is done, perhaps using intact hepatocytes.     

Vitamin B12 Binding Proteins in Liver Disease

Vitamin B₁₂ binding proteins were studied in patients with acute and chronic liver disease, and compared with vitamin B₁₂ binders in chronic myeloid leukemia. In acute viral hepatitis marked elevation of serum vitamin B₁₂ was common. Although the serum vitamin B₁₂ rose as high as that found in chronic myeloid leukaemia the unsaturated vitamin B₁₂ binding capacity (UBBC) was markedly elevated in the latter condition, whereas it was decreased in acute liver disease. In cirrhosis a moderate increase of serum vitamin B₁₂ and UBBC was common. Urinary vitamin B₁₂ excretion increased significantly only when the serum vitamin B₁₂ became markedly elevated in hepatitis. As in chronic myeloid leukaemia the alpha-globulin vitamin B₁₂-binder carried the bulk of the elevated serum vitamin B₁₂ in acute liver disease; the beta-globulin vitamin B₁₂-binder was decreased in serum but seemed to be the predominant binder in urine. This suggests that renal loss of beta-globulin binder is greater than renal loss of alpha-globulin binder. Only minor albumin binding of vitamin B₁₂ in liver disease was found in spite of marked elevation of the serum vitamin B₁₂ level. In cirrhosis the serum alpha-globulin binder was often increased and beta-globulin binder decreased. The cause and significance of these findings are discussed. The ability of serum vitamin B₁₂-binders of liver disease to ‘transfer’⁵⁷Co-B₁₂ to tissue was investigated in an in vitro rat liver homogenate system. In a limited study, vitamin B₁₂ uptake appeared to be within normal limits, except for suboptimal uptake from the beta-globulin binder in cirrhotic serum. Poor vitamin B₁₂ uptake from chronic myeloid leukaemia serum (in particular alpha-globulin binder) was confirmed.     

Formation of Transcobalamin II-Vitamin B12 Complex by Guinea-Pig Ileal Mucosa in Organ Culture After in Vivo Incubation with Intrinsic Factor- Vitamin B12

The in vivo incubation of intrinsic factor--[57Co]vitamin B12 in an ileal loop of a guinea-pig followed by in vitro culturing of segments of the ileum for 180 min has been used to study the transepithelial transport of vitamin B12. Analysis of the solubilized supernate of mucosa following the in vivo phase demonstrated that 44% of the [57Co]vitamin B12 was bound to intrinsic factor (IF), 26% was free, and 16% was bound to transcobalamin II (TCII). Following culture, similar analysis demonstrated that 18% of the vitamin was now bound to IF, 49% was free, and 35% ws bound to TCII. In the culture medium, 54% of the [57Co]vitamin B12 was free and 37% was bound to TCII. The formation of TCII-[57Co]vitamin B12 did not occur if homogenized mucosa was incubated with free[57Co]vitamin B12, but it did form in cultures of ileal segments from animals given an excess of unlabelled vitamin to saturate all circulating TCH, and in the medium containing puromycin. Indirect immunofluorescence using chicken anti-TCII demonstrated that TCII was associated with the mucosal cells of both the ileum and jejunum. These studies demonstrate that following transepithelial flux of vitamin B12 through the ileal mucosa, the vitamin becomes coupled to TCII. This coupling requires a structurally intact mucosa and the source of the TCII appears to be the ileal mucosal cell rather than unsaturated TCII circulating in the blood.     

The Binding of Vitamin B12 by Serum Proteins in Normal and B12-Deficient Subjects

Endogenous B₁₂ in normal serum has been shown to be associated with α globulins (Pitney, Beard and Van Loon, 1954; Heinrich and Erdmann-Oehlecker, 1956; Mendelsohn, Watkin, Horbett and Fahey, 1958). When B₁₂ has been added to normal serum, however, most or all of the added B₁₂ has been associated with the β- and α₂-globulins (Miller, 1958). In the present investigation with radioactive ₅₇cobalt cyanocobalamin (B₁₂*) added to serum, it was possible to define at least two B₁₂-binding proteins (BP), a β-globulin, and an α₁ globulin. B₁₂* added to the serum of normal subjects migrated mainly with the β-globulins on electrophoresis, but when added to the serum of B₁₂-deficient subjects, a small fraction of the B₁₂* was also associated with the α₂-globulins.     

The Excretion of Methylmalonic Acid and Succinic Acid in Vitamin B12 and Folate Deficiency

The excretion of methylmalonic acid (MMA) and of succinic acid was measured in 18 control subjects and in 58 patients with vitamin B₁₂ and/or folate deficiency. The 18 control subjects excreted from 0.0 to 3·5 mg. MMA in 24 hours. MMA excretion was raised in 27 of 41 patients (Group I) with conditions associated primarily with B₁₂ deficiency. These 27 patients included 15 patients with Addisonian pernicious anaemia, two patients following total gastrectomy, two patients with anatomical lesions of the small intestine and eight patients following partial gastrectomy, whereas MMA excretion was normal in four patients with atrophic gastritis and in a further 10 patients following partial gastrectomy. In Group I, MMA excretion was raised in all but one of the patients with serum B₁₂ levels less than 100 pg./ml. due to uncomplicated B₁₂ deficiency, but was invariably normal in patients with borderline serum B₁₂ levels (from 100 to 160 pg./ml.). In the patients with serum B₁₂ levels less than 100 pg./ml., with associated iron and/or folate deficiency, the excretion of MMA tended to be lower than in patients with uncomplicated B₁₂ deficiency, and was often normal. MMA excretion was slightly raised in one of 17 patients with megaloblastic anaemia, primarily due to folate deficiency (Group II), although six of them had serum B₁₂ levels less than 100 pg./ml. The patient with a raised MMA excretion needed B₁₂ therapy for a full haematological remission. The 18 control subjects excreted from 2.0 to 12.5 mg. succinic acid in 24 hours. Succinic acid excretion was subnormal in nine and raised in five patients in Group I. These 14 patients all had serum B₁₂ levels less than 100 pg./ml. None of the Group II patients excreted subnormal amounts of succinic acid but three, including two patients with serum B₁₂ levels less than 100 pg./ml., excreted raised amounts of succinic acid.     

Uptake of Vitamin B12 by Phytohaemagglutinin-Transformed Lymphocytes

Phytohaemagglutinin (PHA)-transformed lymphocytes have been used as a model cell system to study the uptake of radioactive vitamin B₁₂ by haemopoietic cells. Both mature granulocytes and PHA-transformed lymphocytes took up more vitamin B₁₂ than mature, non-transformed lymphocytes. Uptake of vitamin B₁₂ by PHA-stimulated lymphocytes was greatest at 48–72 hr of culture, i.e. at about the time or just before the time of peak DNA synthesis.
Vitamin B₁₂ deficient lymphocytes took up significantly less vitamin B₁₂ than normal lymphocytes. Folate deficient lymphocytes took up an average of about 50% more vitamin B₁₂ than normal but the difference was not statistically significant for the numbers tested. Vitamin B₁₂ uptake by PHA-stimulated lymphocytes was related to their rate of RNA synthesis (measured by ³H-uridine uptake) and was closely related to active cytoplasmic protein synthesis since it was rapidly inhibited by puromycin and cycloheximide.     

Absorption of Vitamin B12 by the Guinea-Pig

The gastrointestinal distribution and subcellular localization of radioactivity in the ileum of the guinea-pig was determined 2 hr after oral feeding of physiological doses of cyano-, methyl- and 5' desoxyadenosyl-cobalamin. The gastrointestinal distribution of the three analogues was similar although the uptake of methyl- and adenosylcobalamin by the ileum was less than that of cyanocobalamin. All three analogues were significantly localized in the mitochondrial fraction of the ileal mucosa during absorption.
Guinea-pigs were injected with sodium fluoroacetate, a drug which is thought to interfere with the conversion of cyano- to adenosylcobalamin. Large doses of the drug caused a marked delay in gastrointestinal transit. The subcellular distribution of radioactivity in the ileum was similar in the control group and those pretreated with a small dose (5μg/100 g) of fluoroacetate.
It is concluded that the mitochondrial localization of vitamin B₁₂ during absorption by the ileum plays a fundamental role in the transport of the vitamin but that interconversion of cyano- to adenosylcobalamin is not the reason for this localization.     

Reversible C3 Hypocomplementaemia in Megaloblastic Anaemia due to Vitamin B12 Deficiency

S ummary . Serum C3 and C4 levels have been determined in patients with Addisonian pernicious anaemia (PA) and megaloblastic anaemia due to vitamin B₁₂ deficiency from other causes, before and after treatment, in order to study the interaction between vitamin B₁₂ deficiency and Complement and the role of complement in the pathogenesis of the gastric lesion of PA. C₃ levels are significantly reduced in vitamin B₁₂ deficiency and return to normal on treatment; C₃ levels correlate with the degree of anaemia but not with serum vitamin B₁₂ levels at diagnosis. C₄ levels are normal. These observations suggest that the observed C₃ hypocomplementaenlia is not a consequence of immune mechanisms, but may be due to altered synthesis of C₃ complement component.     

Role of Vitamin B12 in Folate Coenzyme Synthesis

Normal red cells in man were found to contain predominantly folate pentaglutamates with smaller amounts of tetra- and hexapolyglutamates. There was no change in the type of polyglutamate present in red cells from patients with vitamin B12 deficiency and primary folate deficiency. In contrast to the fall in red cell polyglutamate concentration in vitamin B12 deficiency, there was a marked fall in short-chain folates in early folate deficiency (treated non-anaemic epileptics) and a fall in both short chain and long chain polyglutamates in patients with severe folate deficiency and megaloblastic anaemia. These differences in folate distribution within cells exclude a primary failure to transport methylfolate into cells as the lesion in vitamin B12 deficiency. The failure of folate polyglutamate synthesis in ivtamin B12 deficiency arises either from a failure to provide the proper substrate for polyglutamate synthesis or to a direct requirement for vitamin B12 for polyglutamate synthesis.     

The Separation of Free and Bound Vitamin B12

S ummary . The separation of free and bound vitamin B₁₂ was studied using four binders and two forms of vitamin B₁₂ and effecting separation by charcoal adsorption, bag dialysis, ultrafiltration and gel filtration. In charcoal separation systems the results could be significantly affected by a change in the binder, the form of vitamin B₁₂, the type of charcoal, the mass of charcoal or the presence of albumin. In systems with gastric juice and cyanocobalamin one charcoal separation procedure yielded results which were comparable to those obtained by all other procedures but this did not occur with gastric juice and hydroxocobalamin or with other binders and both forms of vitamin B₁₂.
Results obtained using dialysis for separation varied with the membrane employed. Results obtained by gel filtration were significantly different from those obtained by ultrafiltration and those obtained by one ultrafiltration procedure were always significantly different from those yielded by another. Separation methods which give insignificantly different results in one system do not necessarily yield insignificantly different results in a system with a different binder or different form of vitamin B₁₂.     

Inhibitory Effect of Eggs on Vitamin B12 Absorption: Description of a Simple Ovalbumin 57Co-Vitamin B12 Absorption Test

S ummary . Ovalbumin and egg yolks, mixed separately in vitro with radiocyanocobalamin (⁵⁷Co-vitamin B₁₂), were served to normal volunteers in a cooked form. Ovalbumin, and to a lesser degree, egg yolks were observed to inhibit vitamin B₁₂ absorption. This observation explains the rather poor assimilation of vitamin B₁₂ from eggs labelled in vivo with ⁵⁷Co-vitamin B₁₂.
The assimilation of vitamin B₁₂ from the ovalbumin ⁵⁷ Co-vitamin B₁₂ mixture was also measured in patients with structural alterations and functional disorders of the stomach. In contrast to the absorption test using crystalline radio-vitamin B₁₂, this simple food-vitamin B₁₂ absorption test successfully distinguished between the controls and subjects who had simple gastric achlorhydria and patients who had undergone a Billroth I or II operation or a successful vagotomy with pyloroplasty. Furthermore, this ovalbumin ⁵⁷Co-vitamin B₁₂ test successfully separated patients with pernicious anaemia from those with simple gastric achlorhydria. The ovalbumin ⁵⁷Co-vitamin B₁₂ test, in contrast to that using crystalline ⁵⁷Co-vitamin B₁₂, showed a positive correlation with the serum vitamin B₁₂ concentration. This ovalbumin⁵⁷ Co-vitamin B₁₂ test, being more physiologic and informative than the method using crystalline radio-vitamin B₁₂, is the first simple model for food-vitamin B₁₂ absorption to be proposed for research and for clinical purposes.