Pernicious anemia.

Cobalamin, commonly referred to as vitamin [B.sub.12], is one of the essential vitamins that are obtained only from the diet. The delivery of cobalamin from food to tissues involves first the binding of cobalamin from the diet to the salivary protein haptocorin. In the duodenum, cobalamin is released from the complex by proteolysis. The free cobalamin is then bound to the intrinsic factor in the proximal ileum. The intrinsic-factor cobalamin complex enters the intestinal mucosal cells by receptor-mediated endoycytosis where cobalamin is released, which then binds to transcobalamin. This transcobalmin-cobalamin complex is released into the circulation and from there enters the cells by receptor-mediated endocytosis. In the cells, the cobalamin is dissociated from its transport protein and acts as a coenzyme for methionine synthase and methyl-malonyl-CoA mutase. These enzymes are involved in DNA synthesis and energy production, respectively. The lack of coenzyme methyl-cobalamin leads to the accumulation of homocysteine in circulation, which has been associated with the increased potentiality of heart disease and deterioration of the arteries and nerves. Cobalamin deficiency is present in 1% to 3% of population; 10% to 30% of population above 60 years of age has cobalamin deficiency, and it is expected that the number of persons with vitamin [B.sub.12] deficiency will increase in the next 20 years. Cobalamin deficiency may result from dietary lack of vitamin [B.sub.12], malabsorbtion, and infections. Some of the clinical manifestations associated with vitamin [B.sub.12] deficiency are 1) Hematologic: megaloblastic anemia and pancytopenia (leucopenia, thrombocytopenia); 2) Neurologic: paresthesias, peripheral neuropathy, and combined systems disease (demyelination of dorsal columns and corticospinal tract); 3) Psychiatric: irritability; personality change; mild memory impairment, dementia; depression; and psychosis; and 4) Cardiovascular: possible increased risk of myocardial infarction and stroke.

What is pernicious anemia?

Pernicious anemia is one of the most common causes of cobalamin deficiency. Histologically, pernicious anemia is characterized by gastric mucosal atrophy, selective loss of parietal and chief cells from the gastric mucosa, and submucosal lymphocytic infiltrate. Immunologically, the hallmark of pernicious anemia is the presence of autoantibodies to gastric parietal cells (AGPA), proton pump (H+K+ATPase), and to the cobalamin-absorbing protein, intrinsic factor.

Autoantibodies in pernicious anemia

AGPA are detected either by immunofluorescence on mouse-stomach sections or by ELISA. The identification of H+K+ATPase as the antigen to which AGPA react has allowed the detection of AGPA by ELISA. H+K+ATPase is a membrane antigen with two alpha and beta subunits. AGPA react to both the subunits of H+K+ATPase. AGPA are a marker of autoimmune gastritis whereas intrinsic factor antibodies are associated with pernicious anemia. The progression of chronic atrophic gastritis to pernicious anemia may occur over time and may take 20 to 30 years before this may happen. Thus, the detection of both of these types of antibodies is important, and it imparts greater confidence in the diagnosis of pernicious anemia. AGPA occur in about 90% of patients with pernicious anemia, 30% of first-degree relatives, and up to 50% of adults and 18% of children with Helicobacter pylori infection. In addition, there is high incidence of AGPA in patients with various autoimmune endocrinopathies. In normal subjects, there seems to be an age-related increase in the incidence of AGPA from 2% to 8%. Also, there is a general increase in the number of persons with atrophic gastritis with an increase in age.

Two types of chronic gastritis have been described. Type 1 is considered to be autoimmune whereas type 2 has been associated with H pylori infection. Recent studies, however, have shown that many H pylori infected patients have AGPA and that pernicious anemia earlier considered not to be associated with H pylori infection is presently believed to be H pylori infection associated. About 65% of patients with gastritis and H pylori infection are AGPA positive. The presence of AGPA in such cases is associated with the duration of H pylori infection and the degree of lymphocytic infiltrate and atrophy of the glandular epithelium. It is proposed that H pylori-induced inflammation may result in breakdown of the tolerance for self-antigens (H+K+ATPase and other antigens) in genetically predisposed individuals. As a result of continuous inflammation, epitope spreading to H+K+ATPase and other autoantigen takes place with or without H pylori infection. With hypo or achlorhydria resulting in increase in stomach pH and atrophy with loss of parietal cells that harbor H pylori, the patient eventually may be negative for H pylori and thus resemble Type 1 autoimmune gastritis.

What is an intrinsic factor?

Intrinsic factor is a 60 kD glycoprotein produced by the parietal cells of the stomach lining and enables the absorption of vitamin [B.sub.12]. In acquired pernicious anemia, there is a significant decrease in intrinsic-factor expression due to the loss of intrinsic-factor-producing gastric parietal cells, which results in the body's inability to absorb vitamin [B.sub.12] in the stomach. Approximately 2% of the population of ages >60 have undiagnosed pernicious anemia.

Intrinsic-factor antibodies are of IgG isotype and occur in about 70% of patients with pernicious anemia; they are classified into two types: Type I block the binding of vitamin [B.sub.12] to intrinsic factor and, thereby, prevent the uptake of vitamin [B.sub.12] (blocking antibodies). Type II antibodies bind to a remote site to the blocking antibodies and prevent the attachment of intrinsic factor-cobalamin complex to the ileal receptors (binding antibodies). Both types result in the same pathological effect (i.e. preventing cobalamin absorption). Type II antibodies rarely occur in the absence of type 1 antibodies.

Detection of autoantibodies

Antibodies to intrinsic factor are detected by radioimmunoassay (RIA) or by enzyme linked immunosorbent assay (ELISA). ELISA offers advantages over RIA or other methods that rely on the ability of autoantibodies to inhibit the binding of labeled vitamin [B.sub.12] to intrinsic factor. ELISA detects antibodies of both Type I and II whereas RIA or other inhibiting assays detect only Type I antibodies. RIA and other [B.sub.12] inhibiting antibody methods give false positive results as these assays are subject to interference by serum [B.sub.12] levels. Patients suspected to have pernicious anemia may already be on vitamin [B.sub.12] therapy and the levels of [B.sub.12] in the circulation may be high. Even though ELISA has advantages over RIA, not all ELISA methods of detecting intrinsic factor antibodies may be the same as they relate to the purity of the antigen and the antigenisity of the preparation. Methods employing recombinant glycosylated holoenzyme as the antigen with high degree of purity and consistency provide more reliability in the detection of intrinsic factor antibodies.

Summary

Pernicious anemia is severe anemia most often affecting older adults, caused by failure of the stomach to absorb vitamin [B.sub.12] and characterized by abnormally large red blood cells, gastrointestinal disturbances, and lesions of the spinal cord. Pernicious anemia is caused by a lack of intrinsic factor and could be an autoimmune disorder. The identification of various autoantibodies helps in the confirmation of the diagnosis and hence towards the patient management. In such patients, oral supplements or intramuscular injections of vitamin [B.sub.12] are indicated.

Vijay Kumar, PhD, is degreed in biochemistry with postdoctoral training in clinical immunology. He is board-certified by ABMM and ABMLI. He is CEO and president of IMMLO Diagnostics Inc. (www.immco.com). The company specializes in autoimmune diagnostics. Dr. Kumar has over 30 years of experience in autoimmunology and has published extensively in the field.

By Vijay Kumar, PhD