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Second Edition

PATHOPHYSIOLOGY
The Biological Principles of Disease

LLOYD H. SMITH, Jr., M.D.
Professor of Medicine; Associate Dean, University of California, San Francisco, School of Medicine, San Francisco. California

SAMUEL 0. THIER, M.D.
Sterling Professor and Chairman, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut

1985

W B. SAUNDERS COMPANY
Philadelphia, London, Toronto, Mexico City, Rio de Janeiro, Sydney, Tokyo, Hong Kong

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INTERNATIONAL TEXTBOOK OF MEDICINE
General Editors

A. H. SAMIY, M.D.
Professor of Clinical Medicine and Chief of
Division of Medicine, New York Hospital, Cornell Medical Center
New York, New York

LLOYD H. SMITH, JR., M.D.
Professor of Medicine; Associate Dean,
University of California, San Francisco, School of Medicine,
San Francisco, California

JAMES B. WYNGAARDEN, M.D.
Director, National Institutes of Health,
Bethesda, Maryland

VOLUME I
PATHOPHYSlOLOGY
The Biological Principles of Disease

VOLUME II
MEDICAL MICROBIOLOGY AND INFECTIOUS DISEASES

Volumes I and II of the International Textbook of Medicine
have been conceived and written to follow a logical pedagogical approach
and can readily be used in conjunction with the
CECIL TEXTBOOK OF MEDICINE.

Page 194

    Vitamin B12. In 1926, Minot and Murphy observed that it was possible to treat pernicious anemia by feeding raw liver. In 1929, Castle discovered that the intestinal absorption of the anti-pernicious-anemia principle of liver, which he called extrinsic factor (vitamin B12), depended on its first being bound to a factor secreted by the gastric mucosa, which he called intrinsic factor. Further developments included the identification of the chemical structure of vitamin B12 and the elucidation of its mechanism of action.
    Vitamin B12 is synthesized only in certain microorganisms. Animals depend on microbial synthesis for their vitamin B12 supply. Human foods that contain [high levels of] the vitamin are of animal origin (meat, liver, fish, eggs, and milk). The average daily diet in Western countries contains 5 to 30 ~g of vitamin B12, of which 1 to 5 ~g is absorbed. The total body stores of the vitamin in adults range from 2 to 5 mg, of which approximately 1 mg is found in the liver. The daily dietary requirement is about 2 to 3 p.g. Hence, after stopping intake of vitamin B12, or after total gastrectomy, it takes several years to deplete the body stores and for the symptoms of vitamin B12 deficiency to develop.
    The structure of vitamin B12, or cyanocobalamin, was elucidated in 1955 by the x-ray crystallographic analysis of Dorothy Hodgkin. The vitamin B12 molecule (Fig. 16) consists of two main parts: (1) a planar group, a ring structure surrounding the cobalt atom, that resembles the porphyrin ring of heme except for a bond linking two pyrrole rings directly together instead of through a bridging carbon atom-this structure is called a corrin ring; and (2) a nucleotide group containing the base 5,6 dimethylbenzimidazolyl and phos­phorylated ribose esterified with 1 amino, 2 propranol. Finally, a cyanide group is carried by the trivalent cobalt atom, which is also linked to the benzimidazole base. The term vitamin B,2 may be used to describe cyanocobalamin, although it is often used to include other forms of the vitamin in which the -ON is replaced by another side group. In nature, vitamin B12 exists largely as the two forms, methylcobalamin and 5’deoxyadenosylcobalamin. These are labile [easily changed] and are converted to a fourth form of vitamin B12, hydroxyco­balamin. Both cyanocobalamin and hydroxycobalamin are used therapeutically.

*0.5 mg/dl bloodI24 hours.

    Dietary vitamin B12 is released from protein and peptide complexes in the stomach, where it attaches to both intrinsic factor and a second vitamin B12 binding protein called R-binder. Intrinsic factor is a glycopro­tein that contains 15 per cent carbohydrate and is secreted by the parietal cells of the body and the fundus of the stomach. One molecule of intrinsic factor binds to one molecule of vitamin B12 in the region of the edge of the corrin ring. The R-protein is degraded by pancreatic secretions and the vitamin B12 released binds to further intrinsic factor. This step is impaired in patients with pancreatic disease, leading to reduced vitamin B12 absorption. The normal site of absorption appears to be the distal ileum. It is possible to absorb up to 1.5 pg of vitamin B12 from a single oral dose of intrinsic factor-vitamin B12 complex. Following absorption, the ileum is reffactory [resistant] to further vitamin B12 absorption for several hours. At a neutral pH and in the presence of talcium ions, intrinsic factor-vitamin B12 complex passively attaches to receptor sites on the brush borders of the ileal mucosa. It then enters the mucosal cell, where it is found in the cytosol. After exit from the ileal enterocyte, vitamin B12 is attached to a second major vitamin B12 transport protein (trans­cobalamin II, see below) which is synthesized in the ileal cells. Thereafter, the vitamin is transferred to the portal blood, the peak blood level being reached only 8 to 12 hours after an oral dose. Intrinsic factor does not enter the portal blood and its fate is not absolutely clear. Only about 1 per cent of an oral dose of the order of 30 to 300 p.g of vitamin B12 is absorbed in the absence of intrinsic factor. This form of absorption occurs throughout the gut by simple passive means.
    Vitamin B12 is transported on specific binding proteins called transcobalamins (TC), of which three forms, TOI, II, and III, have been isolated. TOII is the main delivery protein that transports cobalamins to tissues such as marrow and in the nervous system. It is synthesized by a variety of cells, including macro­phages, ileal enterocytes, and the liver. The TOIl­vitamin B12 complex attaches to a receptor site on the surface of various target cells and is internalized by pinocytosis. TOII is essential for vitamin uptake by cells, and in its absence a severe vitamin B12 deficiency state develops. The functions of TO’s I and III, which

are probably produced by leukocytes, are unknown. The TO levels vary in a variety of disease states. As might be expected, TOI levels rise markedly in various forms of granulocytic leukemia and other myeloprolif­erative diseases and also during the leukocytosis of infection. Increased TOII levels are observed in liver disease and pregnancy.
     The metabolic functions of vitamin B12 are only understood in three well-defined reactions. One is the conversion of homocysteine to methionine, as shown in Figure 17. The enzyme involved is homocysteine­methionine methyl transferase, and the reaction requires 5-methyl tetrahydrofolate as a methyl donor, S. adenosylmethionine, and the reducing agent (FADH2) as well as methyl-B12 as a coenzyme. Vitamin B12-dependent methionine synthesis is important in the regeneration of tetrahydrofolate from methyltetrahy­drofolate. The second reaction involving vitamin B12, as deoxyadenosylcobalamin, is the conversion of pro­prionate to succinate, as shown in Figure 18. This reaction is part of the route by which cholesterol and odd-chain fatty acids as well as a number of amino acids and thymine are used for energy requirements via the Krebs cycle. Patients with vitamin B12 deficiency may excrete excess methylmalonic acid. Finally, vitamin B22 is involved in the i~omerization of j3-leucine to ct-leucine; in vitamin B12 deficiency 13-leucine accumulates while ct-leucine is decreased. The inter­relationships between vitamin B12 and folate metabolism in the synthesis of DNA are considered later-it is this function of vitamin B12 that undoubtedly accounts for the megaloblastic erythropoiesis and related phenomenon of abnormal DNA synthesis found in vitamin B12 deficiency states. Vitamin B12 is also essential for the function of metabolic pathways in the central nervous system.

CN
k.
p.
CH2
CHa
Co
NH
 I 0-
 CHa I
  P
 0 C’
CH3
FIgure 16. The structure of vitamin B12. (From Hoflbrand, A.V.: In Hardisty, R. M., and Weatherall, D. J. (eds.): Blood and Its Disorders. Oxford, Blackwell Scientific Publications, 1974.)

Valine
 Isolaucine Thymine
Mothionina
Threonina
Odd chain fatty acids
Methylmalonyl-semialdehyde
 H ATP, Mg, H~ CH3 CH2-COSCoA
 t biotin I I 5’ deoxyadenosyl ~12
H3C-~ -COSCoA+ HcO3 -  H3C-¶-COSCOA Mathylmalonyl H~lj~CoSCoA Methylmalonyl   2
    H -CoA     COOH CoA racamase    COON CoA mutase COON
 carboxylase   L-rn.thylmalony/ - CoA  s~iny/ - CoA
 p~ionyI CoA D-niethylmalonyl - CoA
   ~H3
H-C-COON
COOH
methylmalonic acid
Figure 18. The metabolic interactions of propionyl OoA and succinyl OoA and the role of vitamin B12. (From
HofThrand, A. V.: In Blood and Its Disorders. Hardisty, R. M., and Weatherall, D. J. (eds.). Oxford, Blackwell Scientific Publications, 1974.)

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