Vitamin B12 (VitB12) deficiency is quite common in clinical practice and its incidence increases with age [1]. The prevalence of B12 deficiency in the elderly in developed communities has been estimated to be 12% [1-5], while the prevalence in hospitalized elderly is as high as 30%-40% [6].VitB12 deficiency is often overlooked in its early stages due to mild symptoms. However, in addition to causing megaloblastic anemia, VitB12 deficiency can lead to many neuropsychiatric symptoms, affecting the normal life and work of patients and even causing disability; on the contrary, if timely diagnosis is made and early treatment with VitB12 supplementation is given, the progression of the disease can be stopped or even reversed [7]. It is expected that with the increasing aging of the society, the number of VitB12 deficient people will increase. Therefore, research on the diagnosis and treatment of VitB12 deficiency is of great practical significance and should be given sufficient attention.
First, the absorption process of vitamin B12 is complex
VitB12 is the most complex vitamin in terms of its chemical structure, which cannot be synthesized by human body, so its source depends entirely on food (mainly animal protein) supply. VitB12-R protein complex enters the upper part of the small intestine. In the alkaline environment of the small intestine, the complex is dissolved by pancreatic enzymes, allowing VitB12 to free and bind to the internal factor (IF) secreted by the mural cells. A very small percentage (about 1%) of free VitB12 is absorbed into the circulation through diffusion in the small intestine without relying on endocannabinoids, and after entering the circulation, VitB12 binds to its transporters, cobalamin (TC) I, II and III, and is transported to the liver and other tissues of the body through the portal circulation. The amount of TC II is very small (about 10%), but it is the strongest transporter. Subsequently, the VitB12-TC complex is taken up via cell surface TC receptor-mediated endocytosis; subsequently, lysosomal enzymes degrade the TC, freeing VitB12. The free VitB12 is converted to its active forms adenosylcobalamin and methylcobalamin by enzymatic reactions in the mitochondria and cytoplasm, respectively, the latter two acting as cofactors to convert methylmalonyl coenzyme A (CoA) to succinyl CoA and homocysteine (Hcy) to methionine, respectively [2]. Once VitB12 is deficient, the above reactions are blocked, which leads to an increase in methylmalonyl CoA and its hydrolysis product methylmalonic acid (MMA), as well as Hcy levels.
Second, there are many factors that cause VitB12 deficiency
VitB12 deficiency can be caused by any obstacle in the process of VitB12 absorption, and there are generally the following reasons.
1. Insufficient VitB12 in food The daily requirement of VitB12 for adults is about 2 to 5 μg, and usually 5 to 15 μg of B12 can be consumed from food daily, thus fully meeting the need. In addition, the body has a large reservoir of VitB12, especially the liver, which has a reserve of 2-5 mg. Therefore, it takes 5-10 years for a previously healthy person to develop VitB12 deficiency, even if the diet is completely deficient in VitB12, and 2-5 years for a severely malabsorbed person to develop VitB12 deficiency with a good supply of VitB12 [2]. Thus, clinical VitB12 deficiency due to malnutrition is rare, less than 5% even in the elderly population; it is usually seen only in long-term vegetarians with malabsorption (e.g., alcoholism) [2]. Therefore, malnutrition is not the main cause of VitB12 deficiency.
2, food VitB12 malabsorption Malabsorption of VitB12 in food is the primary cause of VitB12 deficiency in the elderly, accounting for about 60% to 70%. The main cause is the lack of gastric acid due to atrophic gastritis, which prevents the dissociation of VitB12 from food proteins, resulting in a reduction of absorbed free VitB12 [2]. It is estimated that about 40% of elderly people over 80 years of age suffer from atrophic gastritis, many of which are associated with chronic H. pylori infection, thus predisposing them to VitB12 deficiency. Other factors leading to VitB12 malabsorption are numerous, but rare except for endogastric factor deficiency (see below), such as subtotal gastrectomy (which causes gastric acid deficiency), pancreatitis (which prevents VitB12-R protein complexes from being solubilized in the small intestine), intestinal microbial colonization or tapeworm infection (which depletes VitB12), long-term use of biguanide hypoglycemic agents or H2 receptor antagonists and proton pump inhibiting acid preparations (affecting VitB12 dissociation from food), lesions of the terminal mucosa of the small intestine (impairing VitB12 absorption, such as Crohn’s disease, lymphoma, tuberculosis, amyloidosis, scleroderma, Whipple’s disease, aprotininemia, celiac disease, etc.), and other diseases causing impaired VitB12 absorption (such as HIV infection, multiple sclerosis, etc.) [2,8 ].
3, Pernicious anemia Pernicious anemia is the classic cause of VitB12 deficiency and accounts for 10% to 20% of VitB12 deficiency in the elderly. It is an autoimmune disease, mainly mediated by cellular immunity, causing destruction of the gastric mucosa, especially the fundic mucosa, resulting in end-factor deficiency and causing disease; patients also have mural cell antibodies and end-factor antibodies, which dysfunction the α and β subunits of H+/K+-ATPase and end-factor, respectively [9,10]. The sensitivity of detection of mural cell antibodies in pernicious anemia is as high as 85% to 90%, but the specificity is 50%, while the specificity of endokine antibodies is as high as 98% despite a sensitivity of only 50%, so a positive test is a reliable diagnostic indicator. In addition, patients with feedback gastrinopathy have a sensitivity of >80% and a specificity of <50% [7].
Pernicious anemia often coexists with other autoimmune diseases, such as hypothyroidism, Addison’s disease and dry syndrome, and the incidence of tumors (e.g., gastric cancer and lymphoma) is increased. Therefore, patients with pernicious anemia should undergo regular (every 3-5 years) endoscopy with multiple biopsies for early detection of malignancies [2].
4. Hereditary disorders of VitB12 metabolism Certain genetic diseases or VitB12 deficiency due to lack of cobalamin transporting protein II in the body, which makes VitB12 absorption impaired, or due to intracellular lack of enzymes necessary for VitB12 metabolism. These disorders are usually seen in neonates and are rare in the elderly [2,9].
5. Other causes Nitrous oxide, an anesthetic agent, can oxidize VitB12 to reduce it and cause inactivation of methionine synthase. Routine surgery does not usually cause VitB12 deficiency symptoms because of the small amount and short duration of inhaled nitrous oxide; however, prolonged exposure or short duration of large amounts of inhalation can lead to severe neurological symptoms [9]. In addition, those with low VitB12 reserves have a significantly higher chance of developing neurological symptoms after surgery.
Third, VitB12 deficiency is an important cause of neurological dysfunction
VitB12 deficiency impairs neurological function through several mechanisms. First, as mentioned earlier, VitB12 mediates two important enzymatic reactions in vivo. One is the conversion of methylmalonyl CoA to succinyl CoA, and the other is the conversion of Hcy to methionine. In the latter reaction, methylcobalamin acts as a cofactor for methionine synthase, transferring the methyl group of 5-methyltetrahydrofolate to Hcy to enable the formation of methionine. Adenosylated methionine (S-adenosylmethionine, SAM) acts as a donor of methyl groups and is involved in the methylation of many important substances, including DNA, RNA, proteins, myelin and many neurotransmitters. Once VitB12 is deficient in the body, SAM production is blocked, leading to severe metabolic disorders and causing neuropathy such as impaired formation and loss of neuromyelin. Secondly, the accumulation of Hcy due to impaired conversion produces cytotoxic effects through mechanisms such as stimulation of NMDA receptors and activation of apoptosis-related proteins bax and p53, which impair neurological function. Third, impaired methionine synthesis prevents the conversion of methyltetrahydrofolate to tetrahydrofolate, resulting in decreased production of methylenetetrahydrofolate. The latter is an important cofactor for the conversion of RNA precursor deoxyuridine (α-UMP) to DNA precursor deoxythymidine (α-TMP), and its reduction causes blockage of DNA synthesis and neurological dysfunction, which is the so-called “folate trap” hypothesis. Finally, VitB12 deficiency causes the accumulation of S-adenosylhomocysteine (SAH), a powerful inhibitor of many methyltransferases, and its reduced amount inevitably leads to the blockage of methylation process and affects the normal metabolism of neuronal cells [10].
Fourth, VitB12 deficiency neuropathy has a wide range of symptoms
VitB12 deficiency often causes neurological dysfunction with a wide range of manifestations, varying in severity, and in about 40% of cases lacking “classic” symptoms such as anemia [11,12]. Neurological pathologies are quite extensive and include (1) central nervous system, such as dementia, Parkinson’s syndrome and cerebrovascular disease; (2) psychiatric symptoms, such as depression, schizophrenia-like symptoms and mania (megaloblastic mania); (3) spinal cord, such as subacute combined spinal cord degeneration, ataxia and spastic gait; (4) peripheral nervous system, such as sensory polyneuropathy, sensory-motor polyneuropathy, cerebral neuropathies (in the form of anaemia); and (5) neuropathy. neuropathies, cerebral neuropathies (mostly optic and olfactory nerves), and autonomic neuropathies (mainly affecting urinary and fecal functions); (5) myelopathies combined with neuropathies [13]. As can be seen, the symptoms of VitB12 deficiency cover almost all neurological functions and are far from being limited to the classical subacute combined spinal cord degeneration.
V. Diagnostic methods of VitB12 deficiency should be insightful
Clinically, anyone who encounters anemia, has VitB12-related neuropsychiatric symptoms, has a history of gastrointestinal surgery, long-term application of drugs such as glucose-lowering or acid-forming drugs and alcoholism or vegetarianism, especially in the elderly, should be alerted to the possibility of VitB12 deficiency and have their peripheral blood and serum VitB12 concentrations routinely tested [2,7,14]. Peripheral blood picture commonly shows macrocytic anemia with a cell volume (MCV) >100 fl, which often coincides with the degree of B12 deficiency; neutrophil lobulation is excessive and may also present as a whole blood cytopenia; sometimes young red blood cells may be seen. It should be noted that VitB12 deficiency is not necessarily associated with anemia, and even 19-28% of those due to pernicious anemia do not show anemia [9]. The normal level of serum VitB12 is 148-443 pmol/L (200-600 pg/L), and VitB12 deficiency should be considered if it is below 148 pmol/L on two separate occasions. However, a normal level cannot be completely excluded and should be considered in conjunction with clinical symptoms. Studies have shown that 5% to 10% of B12 deficient individuals have serum VitB12 levels above 148 pmol/L; for those with typical clinical features, even if VitB12 is at the low limit of normal levels, as long as they respond well to VitB12 therapy, they should still be considered as having VitB12 deficiency [9]. Bacterial overgrowth in the gut can produce biologically inactive VitB12 analogs, which may create the illusion of normal serum VitB12 levels [2,14].
As previously mentioned, VitB12 deficiency often has a corresponding metabolic impairment, and increased levels of MMA and total homocysteine (tHcy) are sensitive indicators [4,7,15,16]. the normal range for MMA is 80-560 nmoles/L and for tHcy is 5-15 μmol/L. It is usually assumed that VitB12 deficiency is associated with MMA >300 nmol/L and tHcy >15 μmol/L. The sensitivity of both is high, while the specificity of MMA is superior. In patients with VitB12 deficiency, the positive rates of increased MMA and tHcy were reported to be 98.4% and 95.9%, respectively, and 99.8% if both were detected simultaneously. levels and an increase in erythrocyte pressure volume, suggesting that MMA and tHcy are sensitive indicators of early VitB12 deficiency in tissues [5]. If reduced MMA and tHcy levels, as well as the return to normal after VitB12 treatment, are also used as diagnostic criteria for VitB12 deficiency, serum VitB12 levels exceed 200 pmol/L in nearly half of the cases, i.e., if only reduced serum VitB12 levels are used as diagnostic criteria, half of the cases will be missed. The rate of underdiagnosis has been reported to be 10% to 26%. Therefore, for those who are suspected of having serum VitB12 deficiency and whose serum VitB12 levels are at the low end of normal, the presence of VitB12 deficiency can be determined by testing the blood for these two metabolites; if the levels are elevated, this can be confirmed by experimental treatment with VitB12.
It should be noted that about 1/3 of folic acid deficient individuals may show the appearance of VitB12 deficiency with an increase in tHcy. Thus, patients with reduced VitB12 levels or increased tHcy should be tested for folic acid levels promptly. In addition, renal dysfunction and VitB6 deficiency can also be secondary to increased tHcy. Based on the above, Andres et al. extended the diagnostic criteria for VitB12 deficiency to include a serum VitB12 level <150 pmol/L and a serum MMA level >0.4µmol/L or a tHcy level >13µmol/L, based on the exclusion of renal insufficiency and folic acid or VitB6 deficiency, to diagnose VitB12 deficiency [2].
Detection of serum mural cell antibodies and endokine antibodies helps to diagnose pernicious anemia. The Schilling (Schilling) test was once used as an important tool to diagnose endokine deficiency VitB12 deficiency, but it is rarely used nowadays because of the drawbacks such as complicated operation, the need to apply radiolabeled VitB12, and the fact that the test results are affected by renal function. Although the specificity of gastrin level for the diagnosis of pernicious anemia is not high (50%), the sensitivity is more than 80%, which is still a simple diagnostic tool [7]. Bone marrow examination is usually of little significance; MR applied to spinal cord involvement can show abnormal high signal at the site of the lesion under T2W, with the posterior cord of the cervical segment predominating; nerve conduction velocity (NCV) examination can reveal evidence of motor and sensory axonal disease [17].
VI. Awareness of subclinical VitB12 deficiency should be given high priority
Subclinical VitB12 deficiency refers to those with reduced serum levels of VitB12 but without anemia or neurological signs and symptoms. In recent years, there has been an increasing international focus on subclinical VitB12 deficiency, recognizing that most of the reduced VitB12 levels in the elderly are not due to physiological aging, but rather suggest a state of subclinical VitB12 deficiency. reduced VitB12 levels are often accompanied by increased MMA and tHcy levels indicating abnormal VitB12 metabolism at the cellular level [9]. However, even so, many questions remain to be addressed. For example, although subclinical VitB12 deficiency is known to be widespread, the natural course of the disease is superficially understood because it is clinically asymptomatic; although it is likely that all patients with clinical symptoms of VitB12 deficiency must have had a subclinical deficiency phase in the past, the possibility of fluctuation or even reversal of the disease in patients with subclinical deficiency, in addition to its continuous progression, remains elusive.
However, for patients with subclinical VitB12 deficiency, the possibility of fluctuation or even reversal of the disease, in addition to its continuous progression, has not been elucidated. It is undoubtedly costly to conduct blood VitB12 screening in a large population in order to detect subclinical VitB12 deficiency; however, many of these patients may be prevented from developing severe symptoms by treatment, and it is difficult to decide how to weigh the pros and cons. Furthermore, it is not reasonable to diagnose subclinical B12 deficiency based on reduced serum B12 levels alone. Since a reduced serum VitB12 level does not exactly match a VitB12 deficiency at the cellular level, a VitB12 deficiency in tissues cannot be completely ruled out on the basis of a low serum VitB12 level alone. Recently, the detection of levels of the VitB12-associated metabolites MMA and tHcy has been used as an important indicator to assess VitB12 metabolic disorders. However, there are many factors affecting these two metabolites, especially tHcy. In addition to VitB12 deficiency, folic acid or vitamin VitB6 deficiency, renal dysfunction, hypothyroidism, and improper handling of blood specimens can all cause an increase in tHcy, so the results should be judged with caution. Reduced levels of transport cobalamin (TC) II are a sensitive indicator of vitamin VitB12 deficiency, but this test is not yet routinely performed [18]. The deoxyguanosine inhibition test is perhaps the most sensitive method to detect VitB12 deficiency, providing evidence of abnormal intracellular DNA metabolism, even if VitB12 levels are not low; results turn negative with VitB12 supplementation [19]. It should be noted that 20% to 40% of older adults have lower than normal serum VitB12 levels without evidence of metabolic impairment, and thus are not strictly speaking VitB12 deficient. Therefore, the issue of diagnosing subclinical VitB12 deficiency remains to be further explored.
To date, the treatment of subclinical VitB12 deficiency has been inconclusive, with the following generalizations: (1) No treatment for low VitB12 levels without obvious clinical symptoms, which has the advantage of eliminating the cost of universal treatment. (2) Treating all older adults with reduced VitB12 blood levels, an approach that can benefit many people. (3) VitB12 supplementation in all elderly people, regardless of their VitB12 levels, may benefit some people without the cost of VitB12 screening, but it is not known whether there are disadvantages for people with normal VitB12 levels.
VII. VitB12 deficiency requires lifelong treatment
There is no doubt that patients with symptomatic VitB12 deficiency should be treated promptly. Since the cause of VitB12 deficiency is difficult to remove completely, treatment should theoretically be maintained throughout life [2]. In the past, since there was no shortage of patients with impaired gastrointestinal absorption, the classical treatment was VitB12 supplementation by parenteral route, usually intramuscular injection of VitB12 1000 µg once daily; after 1 to 2 weeks, it was changed to 1000 µg once a week; after 1 month, it was changed to once a month for the rest of life. However, since a small amount (1%) of VitB12 can be absorbed in the small intestine by passive diffusion into the circulation, it is presumed that an adequate oral dose (1000 to 2000 µg daily) of VitB12 should be able to achieve VitB12 supplementation and has been confirmed by a number of studies [11,14]. Since low folic acid levels can also reduce VitB12 levels, those with reduced folic acid levels should be supplemented with folic acid first; if VitB12 levels are still low after treatment, VitB12 should be supplemented again.
In conclusion, VitB12 deficiency is a common disease, especially in the elderly. It has many causative factors, and its damage to the nervous system is extensive and harmful. The diagnosis of VitB12 deficiency is a complex and meticulous process, and clinical vigilance should be enhanced to improve the level of diagnosis, so that patients and subclinical deficiencies can be detected early and treated accordingly to prevent or reduce neuropathy. In addition, there are still many issues to be further explored in the diagnosis and treatment of VitB12 deficiency, especially in subclinical deficiency.