Laboratory Investigation of Vitamin B 12

Transcription

1 Laboratory Investigation of Vitamin Deficiency Majid Moridani, PharmD, PhD, DClinChem, 1,2 Shana Ben-Poorat, BS 1 ( 1 Department of Pharmaceutical Sciences, School of Pharmacy, 2 Department of Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Amarillo, TX) DOI: /CVHKLE2R4W68K2NQ Received Revisions Received Accepted Abstract Vitamin deficiency is not uncommon, particularly in certain patient populations such as elderly, neuropsychiatric, and psychiatric patients. Historically, presentation of vitamin deficiency was recognized as megaloblastic anemia, which is now rarely seen in Western society. Hematological changes, including macrocytic anemia, are a more well-known symptom, but are not always present or are revealed later in the disease course. However, the advances over the last few decades, from the use of the obsolete Schilling test and total serum vitamin level to newer methods such as holo-transcobalamin II, the metabolites methylmalonic acid and homocysteine, antibody testing, and gastrin levels, have created a more accurate diagnosis of the disease. Total serum vitamin level, though sometimes misleading, remains a mainstay of clinical diagnosis. In this work, we will discuss vitamin deficiency, various diagnostic tests, and an algorithm used in the investigation of vitamin Presentation of vitamin deficiency was historically recognized as megaloblastic anemia. Awareness of pernicious anemia as a gastrointestinal disorder dates back to the 1800s. 1 The 19th century also witnessed the first description of neurological abnormalities, including sensory changes, in pernicious anemia. With the link between vitamin and pernicious anemia elucidated, the now infamous Schilling test was developed in the 20th century to test vitamin absorption. 2 The diagnosis of vitamin deficiency is complicated; however, it is important to make a distinction between sub-clinical and clinical deficiency in the clinical setting. Mild, sub-clinical deficiency is particularly common in the elderly and is generally responsive to vitamin therapy. Vitamin The 1934 Nobel Prize in Physiology and Medicine was awarded to Minot and Murphy for the discovery that consumption of liver or injection of a liver extract, later found to include gastric intrinsic factor, was able to cure pernicious anemia. 3 Around the 1950s, vitamin was finally isolated and crystallized. Its structure was determined by x-ray diffraction, a major feat for such a large molecule, winning the 1964 Nobel Prize in Chemistry for Dorothy Hodgkin. 3 Chemical synthesis of the vitamin occurred in The cobalamin family includes several molecules with a central cobalt atom, surrounded by a tetrapyrrole ring similar to chlorophyll or porphyrin. This core of vitamin is often called a corrinoid or corrin ring. 4 The cobalt atom is additionally attached to a nucleotide (5,6 -di-methylbenzimidazole ribonucleotide) and another group that determines its name. Attachment of cyanide as the sixth ligand makes cyanocobalamin (vitamin ), the common form of the drug used in the United States. 5 Attachment of a hydroxyl group yields hydroxocobalamin (vitamin a), the form found in food, the common drug form used in Europe, and a cyanide antidote. However, cells can only use cobalamin in the form of methylcobalamin and deoxyadenosylcobalamin, which are synthesized intracellularly from hydroxocobalamin as described in the section on intra-cellular metabolism. Physiology Absorption, Transport, and Storage Vitamin, synthesized by bacteria only, is present in dietary animal protein sources but not in vegetables. 6,7 Common sources of vitamin are meat, poultry, fish, cheese, eggs, and fortified cereals. Because many older people may be unable to absorb naturally occuring vitamin, people older than the age of 50 are advised by the United States Department of Agriculture to obtain their dietary intake of vitamin from fortified foods or a supplement. 8 The adult recommended daily allowance is 2.4 µg. The average daily diet contains 3 to 30 µg with 2 to 3 µg absorbed by the GI tract. To initiate its absorption, vitamin must be released from food, a process accomplished by gastric hydrochloric acid and pepsin (cleaved from pepsinogen by hydrochloric acid). Confusing nomenclature comes into play when describing the many cobalamin binding proteins (CBPs). Generally, cobalophilin, haptocorrin (HC), and R-binder are used synonymously and include transcobalamin I and III (TC I, TC III). The well distinguished CBPs are transcobalamin II (TC II) and intrinsic factor (IF), both physiologically important in vitamin transport. For an extensive explanation of this nomenclature and some suggested terminology see Markle s review. 4 Upon ingestion, cobalophilin is the first protein to bind vitamin, in the saliva and gastric secretions. Intrinsic factor, Downloaded 166 from LABMEDICINE Volume Number 3 March 2006 labmedicine.com

2 a glycoprotein secreted by parietal cells in the stomach, binds vitamin as it passes through the duodenum of the small intestine, in conjunction with its release from cobalophilin by pancreatic protease (Figure 1). Then, the vitamin -intrinsic factor complex (Cbl-IF) binds its receptor, cubilin, in the distal ileum to allow transport into mucosal epithelial cells. Even without intrinsic factor a small amount of vitamin (about 1%) 9 can still pass into the circulation by passively diffusing through the ileum, justifying the use of mega-doses of oral vitamin (1,000 µg) for patients lacking intrinsic factor. 9 Next, cathepsin L degrades intrinsic factor in the enterocytes. 10 Transcobalamin II from these same ileal cells transports vitamin via capillaries toward the hepatic portal vein and into the entire bloodstream. Several years supply of vitamin, 2 to 3 mg, can be stored in the liver and excreted into the bloodstream as needed, making deficiency by lack of dietary intake rare. Any excess is excreted renally. Also, substantial enterohepatic cycling occurs: 3 to 10 µg of vitamin are excreted in the bile daily, 9 about half of which is reabsorbed by the intestines. 11 The shortlived complex of transcobalamin and vitamin moves from the circulation to target cells where it is endocytosed via the transcobalamin receptor. 12 The number of TC receptors on the cell surface is dependent on tissue requirements. 13 The transfer of vitamin from one binding protein to another is thought to be a defense mechanism to protect vitamin from use by gastrointestinal bacteria 10 and from photodegradation 4 to spare it for use by the body. Vitamin absorption, transport, and storage are exhibited in Figure 1. Intracellular Metabolism Figures 2 and 3 show the metabolic alterations of vitamin that precede its use as a cofactor, most notably the conversion of cyanocobalamin to hydroxocobalamin and then to its active forms, methylcobalamin and adenosylcobalamin. 14 The 2 main reactions requiring vitamin are the conversion of methylmalonyl-coa to succinyl-coa in mitochondria by the enzyme methylmalonyl-coa mutase and adenosylcobalamin, and secondly, the cytoplasmic conversion of homocysteine to methionine by methionine synthase and methylcobalamin. In addition to synthesizing methionine, the formation of methylcobalamin removes the methyl group from 5-methyl-tetrahydrofolate yielding tetrahydrofolate, a process necessary to synthesize purine nucleotides and thymidilic acid for DNA replication. Constant DNA replication makes hematopoiesis possible. A third, less mentioned reaction involving vitamin is the isomerization of α-leucine to β-leucine by leucine 2,3-aminomutase. 4 Pathophysiology of Vitamin B12 Fifty years ago, vitamin researcher Castle said of pernicious anemia, this disease would not develop if the patient could effect daily the transfer of a millionth of a gram of vitamin the distance of a small fraction of a millimeter across the intestinal mucosa and into the bloodstream. 15 Pernicious anemia (PA) and food-cobalamin malabsorption are the 2 most common causes of vitamin deficit, both with gastrointestinal etiology. 9 Some other possible causes of vitamin deficiency are summarized in Table 1. Pernicious anemia does not require the manifestation of anemia in a hematological sense. Rather, it signifies that gastritis and a lack of intrinsic factor prevent intestinal uptake of vitamin. 1,16 The lack of intrinsic factor is due to auto-antibodies to either parietal cells (anti-pc) or intrinsic Figure 1_Vitamin absorption, storage and transport. Vitamin is liberated from food as hydroxocobalamin (OH-Cbl) or from a supplement as cyanocobalamin (CN-Cbl). These forms or vitamin bind intrinsic factor, which binds cubilin, the intestinal IF receptor. Before entering the blood circulation it releases from IF and binds transcobalamin II, forming holo-transcobalamin II (holo-tc II). Upon entering the liver it can be stored or continue though the circulation, allowing binding by tissue-bound transcobalamin receptors. The resultant uptake of holo-tc II into tissues permits vitamin utilization by cells. Figure 2_Intracellular inter-conversion of vitamin. Following receptor-mediated endocytosis of holo-tc II by the transcobalamin receptor, holo-tc II dissociates in a lysosome and free vitamin is able to convert between forms. Cyanocobalamin (CNCbl) converts to hydroxocobalamin (OHCbl) by cobalamin reductase. Hydroxocobalamin can subsequently take several paths. It may be modified to 5'-deoxyadenosylcobalamin (AdoCbl) in the mitochondria and encounter methylmalonyl-coa mutase (Mut). Hydroxocobalamin can alternately be converted to methylcobalamin (MeCbl) which activates methionine synthase (MS). Another possible reaction is conjugation to glutathione (GSH). Downloaded labmedicine.com from March 2006 Volume 37 Number 3 LABMEDICINE 167

3 factor (anti-if). Antibodies to the proton pumps in parietal cells slowly cause atrophic gastritis so intrinsic factor production slows over several decades. 10,17,18 Since vitamin is not adequately being delivered to the usually proliferative gastrointestinal mucosal cells, exhaustion of tetrahydrofolate hinders DNA synthesis 19 and cell division leading to atrophic gastritis. Broadly speaking, the diminished oxyntic glands of the stomach can no longer secrete hydrochloric acid and pepsinogen required for the release of vitamin from food. 20 The consequent hypochlorhydria, insufficient hydrochloric acid to break down vitamin from food, causes partial food-cobalamin malabsorption and furthers vitamin deficiency at a slow rate. Food-cobalamin malabsorption is often associated with the mild, preclinical deficiency seen in the elderly, 21 due to their inherently lower gastric acid secretion. Hypochlorhydria can also lead to bacterial overgrowth. These gastrointestinal bacteria may bind the available vitamin for their own use, exacerbating the malabsorption. 22 Atrophic gastritis also leads to lack of ability to secrete intrinsic factor creating a more complete loss of vitamin uptake, similar to that which occurs in patients with autoimmune-associated pernicious anemia. Because autoimmune pernicious anemia does not affect the antrum of the stomach, antral pyloric glands still secrete excess gastrin to attenuate the acid deficit. Gastric mucosal atrophy may spare enough intrinsic factor to maintain hematopoiesis while not enough for axonal function, 23 hence some recent evidence of neurological aberrations without any anomalous hematological findings. It is possible that hematopoiesis has priority in the usage of vitamin. Gastritis caused by Helicobacter pylori infection prompts a more severe food-cobalamin malabsorption. It is distinct from atrophic gastritis because pepsinogen levels tend to rise in this more superficial inflammation. 24,25 Several congenital or acquired mutations can prevent vitamin absorption and utilization. For example, cubilin mutations in Finnish subjects and mutations in its chaperone protein, amnionless, in Norwegians are rare autosomal recessive Figure 3_The main metabolic reactions requiring vitamin. Vitamin is used by methylmalonyl-coa mutase and methionine synthase to produce succinyl-coa and methionine, respectively. In the absence of vitamin the levels of methylmalonic acid and homocysteine are elevated. Table 1_Some Causes of Vitamin Deficiency 9 Inadequate ingestion: Veganism, chronic alcoholism, poverty, religious tenets Food-cobalamin malabsorption: Atrophic gastritis, pancreatic exocrine failure, drugs, achlorhydria, H. pylori infection Inadequate intrinsic factor: Antibodies to IF or PC (pernicious anemia), gastrectomy, atrophic gastritis Intestinal malabsorption: Small intestinal disorder (Crohn s disease, sprue), intestinal resection, competition by intestinal parasites or bacteria, Immerslund-Gräsbeck syndrome Inadequate utilization: Vitamin antagonists (nitrous oxide), congenital or acquired intracellular enzyme deficiency or deletion, congenital or acquired abnormal binding to cobalamin binding protein Increased requirements: Pregnancy, breastfeeding, hyperthyroidism, infancy, malignancy disorders causing Imerslund-Gräsbeck syndrome, malabsorption of vitamin in spite of adequate intrinsic factor. 26 Transcobalamin II mutations are rare, they occur most often as the result of autosomal dominant inheritance, and patients with these mutations present with chronic neurological symptoms and late-onset anemia. 27 Masking of vitamin deficiency by exposure to large doses of folate presents further problems in diagnosis and could lead to irreversible neurological damage In this situation, there is a lack of methionine, necessary for neurological maintenance, but sufficient folate for hematopoiesis to continue. Anemia is corrected so a folate deficiency may be assumed. Since much food is now being fortified with folic acid to prevent neural tube defects in fetuses, concern about neurologic aggravation in individuals with low-normal vitamin levels is well-founded. 31 Several drugs are implicated in vitamin Drug-induced achlorhydria is not usually a major risk-factor for vitamin malabsorption and Nevertheless, an exception is the long-term use of proton pump inhibitors (eg, omeprazole), as for Zöllinger-Ellison syndrome, causing prolonged achlorhydria. 32 This can lead to food-cobalamin malabsorption. 15 Pharmacogenetics can also affect predisposition to vitamin Polymorphisms in the CYP2C19 gene, for instance, can lower the rate of metabolism of omeprazole. Prolonged omeprazole therapy (1 year) may lead to lowered serum vitamin levels, most commonly in Asians. 33,34 Metformin interferes with the metabolism of calcium leading to vitamin malabsorption in 10% to 30% of patients on this diabetes drug. 8 Cholestyramine also inhibits absorption from the intestines. 35 Nitrous oxide binds vitamin on the corrin ring, disabling it from acting as a cofactor. Presentation of vitamin deficiency was historically recognized dogmatically as megaloblastic anemia that progresses to a neurological condition designated as subacute combined degeneration. This was typically seen in magnetic resonance imaging as sclerosis of the dorsal spinal cord in adults or cerebral atrophy in infants. 26 Recently, however, the view that anemia precedes neurological problems has changed. Physicians may see neurological or neuropsychiatric symptoms alone, anemia alone, or neurological symptoms prior to onset of anemia. 16 When neurological abnormalities are noticed, irreversible cerebellar and spinal injury may have already occurred. 36 The differential expression may be due to genetic or acquired characteristics. 16 While the etiology of anemia in vitamin deficiency has long Downloaded 168 from LABMEDICINE Volume Number 3 March 2006 labmedicine.com

4 been traced to folate imbalance, changes in metabolic status still remain unclear in neuropathies. 37 We now know of the mild, preclinical vitamin deficiency which may be noticed even years after malabsorption begins. Mild, sub-clinical deficiency is particularly common in the elderly and is generally devoid of megaloblastic anemia, yet consistent with metabolic insufficiency, 16 and responsive to vitamin therapy. Peripheral neuropathies are common early neurological symptoms, and wide-ranging mental indications have been shown to result from vitamin deficiency as well. 4,27 Digestive manifestations are few but classic, and several gynecological problems have been under study. 9 Vitamin deficiency may present differently in children than adults. Specific symptoms are listed in Table 2. Clinical Investigation Because vitamin deficiency is not uncommon, particularly in certain patient populations such as the elderly wherein more than 20% are affected, 9 effective identification is essential. It is estimated that 800,000 elderly Americans, especially black and white women, have undiagnosed pernicious anemia causing vitamin 28 Food-cobalamin malabsorption accounts for more than 60% of all cases of vitamin deficiency, compared to only 15% to 20% for pernicious anemia. A list of those who should be tested for vitamin deficiency is given in Table 3. Deficiency of vitamin can be best identified and monitored for improvement by sensitive and specific tests. Hematological changes, including macrocytic anemia, are more well-known characteristics, but are not always present or are revealed later in the disease course. 16 The diagnosis of vitamin deficiency is far from straightforward, due to many testing options and a lack of consistent reference ranges. However, the advances over the last few decades from use of the Schilling test, now obsolete, and total serum vitamin level to newer methods such as holo-transcobalamin II, the metabolites methylmalonic acid and homocysteine, and antibody testing have allowed for earlier, more accurate diagnosis. It is important in the clinical setting to make a distinction between sub-clinical and clinical 38 Total serum vitamin level, though sometimes misleading, is a mainstay of clinical diagnosis. 38 A flow chart of a suggested testing sequence following serum vitamin evaluation is given in Figure 4. Table 2_Possible Symptoms of Vitamin Deficiency 9,26,37 Neuromotor symptoms: Paresthesias, weakness or fatigue, muscle ache, ataxia, abnormal gait, decreased reflexes, reduced vibration sense, restless legs syndrome Neuropsychological symptoms: Dementia, depression, mania, psychosis, impaired memory, disorientation, irritability, hallucinations, personality changes, obsessive-compulsive disorder Digestive: Glossitis, jaundice Gynecological: Vaginal atrophy, mycotic infections, miscarriage, lowered fertility More common symptoms in children: Hypotonia, apathy, decreased visual contact, lethargy, coma, involuntary movements Other: Weight loss, hair loss, headache Table 3_Who Should Be Tested for Vitamin Deficiency? 9 Anemia patients with elevated MCV Patients with unexplained mental status changes Patients with neurological or motor symptoms Institutionalized patients Patients older than 65 years old Vegans and pregnant or breast feeding vegetarians Newborn children of vegetarian, malnourished, or pernicious anemia mothers Gastric surgery patients Atrophic gastritis patients H. pylori infected patients Chronic users of H 2 blocking agents, gastric ATPase inhibitors (PPIs), and metformin Thyroid disease, diabetic, and HIV patients Figure 4_Sample algorithm for clinical investigation of vitamin deficiency and pernicious anemia. Downloaded labmedicine.com from March 2006 Volume 37 Number 3 LABMEDICINE 169

5 Individual Tests Hematological Markers Historically, hematological markers were used to diagnose vitamin Today, it is understood that this places patients at risk of not being diagnosed. More specific tests with diagnostic capability earlier in disease progression are preferred. Still, hematological parameters can sometimes be useful in corroborating the conclusion. Because the complete blood count is so commonly ordered, a low red blood cell count is often the first sign that points to vitamin A peripheral blood smear can visually identify abnormal cells. Megaloblastic macrocytic anemia is the classic sign, but often absent in mild cases. 39 Concurrent iron deficiency can eliminate any macrocytosis, further confusing the diagnostic process. 39 Decrease in number with increase in size of red blood cells, as confirmed by low hematocrit and increased mean corpuscular volume (MCV), and increase in hemoglobin content are cardinal microscopic findings. Hyper-segmented neutrophils, observed as the presence of 5 or more nuclear lobes, are common in mild megaloblastic anemia but not exclusive to vitamin 9,29 Schilling Test Now nearly obsolete due to numerous drawbacks, the Schilling test should be avoided. It was designed to reveal and differentiate causes of vitamin deficiency, a part of the diagnosis now being largely ignored. 38 Vitamin was generally injected and followed with a dose of radioactively labeled oral vitamin. The percentage of radioactive vitamin excreted in a 24-hour urine collection was low in malabsorption diseases including pernicious anemia. The second phase of the test involved adding exogenous intrinsic factor (IF) to the mix. If this increased the excretion to normal, pernicious anemia was the culprit. Failure of IF to increase excretion usually indicated other causes of malabsorption such as small intestinal lesion, bacterial overgrowth, or possibly tapeworms. 40 The reference ranges for excretion percentage are not well defined. The cost of a Schilling test is high, including the oral radioactive vitamin, parenteral vitamin, intrinsic factor, equipment depreciation and use, laboratory material, and professional and administrative staff salaries. 41 With the radiological hazards, reduced availability of 57 Co-, and today s alarmingly high medical costs, it is rational to abandon this less cost-effective test. 40 Other problems with the Schilling test are numerous and have led many laboratories to render this test obsolete. In practical terms, ineffectual collection of a complete 24-hour urine sample can lead to lower excretion percentages being recorded, causing false positives for malabsorption. This has been reported as causing deceptive irregularities in up to 14% of patients. 40,42 Often stage II of the Schilling test will remain abnormal even in pernicious anemia (exogenous IF will not correct the malabsorption) because of the fact that pernicious anemiainduced deficiency can beget ileal dysfunction. 40 The standard Schilling test does not detect food-cobalamin malabsorption because such patients can absorb non-food-bound vitamin without difficulty, causing a normal stage I Schilling test. To overcome this, a modified Schilling test was developed, also called the food-cobalamin absorption test. 4 A low excretion in the modified test but not in the standard Schilling test indicates an inability to cleave vitamin from food rather than an inability to absorb it. The modified Schilling test is not practical for the average clinical laboratory and has come under fire for its insensitivity and is auspiciously outdated by more reliable tests. Serum Vitamin Level Serum vitamin level seems an obvious, straightforward way to diagnose vitamin deficiency and monitor the effectiveness of treatment. Indeed, it is a starting point in the clinical laboratory. Three generations of serum vitamin tests have developed during the last 60 years. In the 1950s, microbiological tests were employed as a first generation test. If certain vitamin -dependent bacteria grew adequately when exposed to the patient s body sample, vitamin from the sample was considered ample. This test is now obsolete. In the 1960s, radioisotopic procedures became common using 57 Cocyanocobalamin and a specific binder and measuring the radioactivity. 43 This had the obvious drawback of radiation exposure risk. Current methods include automated non-isotopic procedures using chemiluminescence, the detection of light energy as an indicator of vitamin concentration (Figure 5). Devices such as the ACS:180 and the ACCESS Immunoassay System are benchtop instruments that perform numerous immunoassays including vitamin and RBC folate. 44 Figure 5 shows the basic principles of the vitamin immunoassay. A practical issue with testing serum vitamin levels is the photosensitivity of the sample. The innate instability of the vitamin requires that samples be frozen for transport and protected from light if not able to be assayed within 4 hours. 40,45 The most frequent reason for abnormal high serum vitamin level is renal failure. If TC I or TC III is high, as in myeloproliferative, renal or hepatic diseases, the serum vitamin level in a deficient patient can be normal or even high yet unavailable for transport into vitamin hungry tissues, thus concealing the 11,40 Colonization by intestinal bacteria, which produce vitamin analogues that are measured with active, can also deceptively raise the results. Figure 5_Principle of the Beckman ACCESS Immunoassay System: serum vitamin. To measure vitamin, the ACCESS device combines the sample with cyanide and dithiothretol to convert all vitamin to cyanocobalamin and denature the binding proteins. Paramagnetic particles covered in both goat anti-mouse and monoclonal anti-intrinsic factor antibodies are added along with alkaline phosphatase bound to intrinsic factor. The vitamin in the sample binds the intrinsic factor. This event prevents the intrinsic factor from binding to anti-intrinsic factor solid phase. After paramagnetic particles are separated out by applying a magnetic field, it is washed and treated with a chemiluminescent substrate (Lumi-Phos) that reacts with the alkaline phosphatase enzyme to produce light. The amount of light generated is inversely proportional to the vitamin concentration, which is determined by a calibration curve. Downloaded 170 from LABMEDICINE Volume Number 3 March 2006 labmedicine.com

6 The historical lower reference value for serum vitamin is 200 ng/l (148 pmol/l). There has been some consideration given to raising the low endpoint to around 350 ng/l, but the benefit in catching these deficiencies may be limited, as they are generally subclinical and with significantly fewer metabolic abnormalities. 38 Additionally, it seems that few subclinical deficiencies progress to serious clinical deficiencies. 16 Some benefit could be perceived by the financial resources saved in providing preventive care to these few patients in need of vitamin therapy. As it is now, low-normal levels are often ignored by physicians, potentially threatening the patient s health, and even genuinely low levels may be ignored if no neuropathy is evident. This problem would be further exacerbated by raising the low reference value. A level less than 300 ng/l certainly warrants further testing, using a functional test such as the methylmalonic acid (MMA) assay described below. RBC Folate By measuring the folate in red blood cells only, a measurement of folate that considers body stores is obtained, 26 so it is more indicative of long-term folate availability than serum folate that may be influenced by recent diet. It is several fold higher than serum folate, therefore, its measurement is more reliable. Often it is ordered with a vitamin level when anemia or neuropathy is seen or to monitor treatment effectiveness. This test will reveal low red blood cell folate if vitamin is not available to receive the methyl group from methyltetrahydrofolate to form tetrahydrofolate. This test can be performed by immunochemiluminescence with a framework similar to the vitamin immunoassay. 44 Reference ranges depend on age, gender, test method and other factors. Several sources doubt this test s reliability and reproducibility. 26,30 It has limited specificity for vitamin deficiency, as RBC folate decrease may also be due to folate or vitamin B 6 However, folate deficiency is rare today, mainly due to food fortification with folate. 46 Deoxyuridine Suppression Test (dust) This sensitive test of cellular vitamin deficiency can detect mild, preclinical forms of the disease. 47 Added deoxyuridine will suppress 3 H-thymidine incorporation into DNA in bone marrow cells in the presence of sufficient vitamin. 48 In a deficiency of either vitamin or folate, the suppression is impaired due to lack of tetrahydrofolate. Differentiation of cobalamin deficiency from folate deficiency is possible by incubating the cells with 1 vitamin and retesting for enhancement of the suppression. Often, dust results will show an abnormality even earlier in the progression of vitamin deficiency than the metabolites methylmalonic acid and homocysteine. 48 Suppression of thymidine addition into DNA may also be due to other disease states, but overall the dust has adequate specificity for vitamin 49 This test, developed in the 1960s, is not practical on the whole because of its cost and the invasiveness of collecting a bone marrow sample. 16 Serum Methylmalonic Acid and Plasma Total Homocysteine Methylmalonic acid (MMA) and total homocysteine (thcy) are measurable metabolites that can build up in vitamin These metabolites are usually elevated before serum vitamin is decreased or symptoms express. 50 Quantitative MMA and thcy testing can be performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) stable isotope dilution analysis by a reference laboratory. 51 A random urine specimen after an overnight fast or a fasting blood specimen is used for testing. However, it is generally believed that a blood specimen is more suitable for MMA measurement. Methylmalonic acid may be similarly measured from amniotic fluid as an early test for neural tube defects. 52 Newer thcy assays include enzyme and immunoassays, which are more suitable for the routine laboratory. 53 Used together, MMA and thcy can distinguish vitamin deficiency from folate and vitamin B 6 In most cases, a vitamin B 6 or folate deficiency will only increase thcy, whereas, a vitamin deficiency will increase both thcy and MMA. 40 Most patients that respond to vitamin therapy have elevations of both metabolites. 54 With its inverse relationship with vitamin level, MMA elevation is a sensitive indicator of even mild vitamin 5 -Deoxyadenosylcobalamin (AdoCbl) is the form of vitamin used in the conversion of L-methylmalonyl-CoA to succinyl-coa, an intermediate in the citric acid cycle. A lack of AdoCbl decreases activity of the enzyme methylmalonyl-coa mutase resulting in buildup of L-methylmalonyl-CoA and its precursor D-methylmalonyl-CoA, which is cleaved into coenzyme A and methylmalonic acid by a hydrolase. 55 The increased methylmalonic acid in the blood and urine is then detected. Serum MMA is more sensitive and reliable than urine MMA because the urine level can be affected by diet and sample collection. 40 However, renal insufficiency can falsely raise serum MMA but not urine MMA results. 16 If serum MMA is abnormally high but vitamin level is normal, the cause may be the higher sensitivity or earlier detection ability, or alternatively a fluctuation with time and health status. 40,56 Therefore, a patient with a high MMA level without any clinical symptoms should be investigated further before finalizing the diagnosis. The major drawbacks of the MMA assay are the need for expensive equipment and the cumbersome methodology. Homocysteine (Hcys), often known as total homocysteine (thcy) because of inclusion of the oxidized and reduced forms, is another significant test for vitamin Methylcobalamin (MeCbl) along with folate and vitamin B 6 are essential in the reaction converting homocysteine to methionine. 5-methyl-tetrahydrofolate serves as the carbon donor, converting hydroxocobalamin to methylcobalamin (Figures 2 and 3). Methylcobalamin then donates the methyl group to homocysteine, generating methionine, an amino acid. Lack of any of the vitamins, B 6, or folate will likely cause increased homocysteine levels. This is physiologically important, as hyperhomocysteinemia is known to cause early cardiovascular disease 29,57 and is associated with risks of neural tube defects, 6,58 dementia, 6,59 Alzheimer s disease, 59,60 and osteoporosis. 26 However, depleted vitamin is not necessarily to blame for high homocysteine, which is why this test is well-known as non-specific for vitamin There is not currently any standardization of thcy assays between laboratories, although most assays give similar results. 40 Because a protein-rich meal can skew homocysteine results upward by as much as 10% to 15%, a fasting plasma collection is necessary. Conversely, collections taken from a patient in the supine position can lower results up to 10%. Normal levels can be twice as high in old age as childhood and vary within any given individual by 8%. 40 Renal insufficiency is notorious for instigating hyperhomocysteinemia. 16,61 Common drugs such as statins (HMG-CoA reductase inhibitors) and estrogens reduce thcy while diuretics and levodopa can increase thcy. 40 Refsum and colleagues define the critical factors to take into account when comparing thcy level to reference values as age, sex, pregnancy, lifestyle, diseases, and drugs. 53 They also suggest defined blood sample handling procedures, including centrifugation or cooling of the sample within an hour. Downloaded labmedicine.com from March 2006 Volume 37 Number 3 LABMEDICINE 171

7 Holo-transcobalamin II (Holo-TC II) Holo-transcobalamin II (holo-tc II) is a method that allows the clinician to determine the amount of vitamin directly available to cells. Transcobalamin II is the only protein that transports vitamin into target cells. This test disregards free vitamin and that bound to other haptocorrins because they are not used metabolically and are less significant physiologically. The concept is similar to measuring free thyroxine versus total thyroxine. In one method, vitamin is measured initially, then all transcobalamin II is removed, and vitamin is remeasured. 62 The difference between the 2 vitamin values provides a good estimate of how much vitamin is bound to TC II, giving the holo-tc II level. A newer method measures TC II directly by removing the transcobalamin II from serum via a solid-phase capture technique. 63 Monoclonal antibody specific for human transcobalamin II concentrates the TC II on magnetic microspheres. Vitamin is then released from the TC II and quantified by competitive protein binding radioimmunoassay. It makes use of calibrators of recombinant human transcobalamin. This test has the benefit of a highly concentrated holo-tc II in the measured sample. 62 A kit is commercially available from Axis-Shield. A second recently developed method uses magnetic beads coated with vitamin to precipitate apo-transcobalamin (the fraction of TC II not bound to vitamin ). 64 ELISA (enzyme-linked immunosorbent assay) can then be used to measure only the holo-tc II, present in the supernatant. This avoids the need to measure vitamin. A low holo-tc II result is an early, sensitive marker of vitamin deficiency so it can more easily prevent real clinical deficiencies. 26 It is not useful in diagnosing malabsorption. 65 One study suggested holo-tc II might replace the combination of vitamin level and metabolites (MMA and homocysteine) to test for vitamin 66 Problems with holo-tc II measurement do exist. Because holo-tc II is not only delivered to tissues at varying rates depending on requirement but also cleared and re-released by the liver and kidneys, the efficacy of these 3 sinks can alter the serum holo-tc II concentration. 62 Additionally, renal tubules and ileal cells produce TC II. Therefore, the concentration of holo-tc II depends somewhat on its level of production. A low reference value for holo-tc II is around 40 pmol/l. Since holo-tc II is such as small percentage of all haptocorrins, a small deviation is assigned a large significance, but in reality could have other contributing factors. 26,62 Holo-TC II results are generally increased in renal failure, as are other tests of vitamin deficiency, leading to the possibility of a false negative. Holo-TC II has been shown to be significantly higher in women than in men. 67 While holo-tc II testing gives better sensitivity than MMA or homocysteine, 26 widespread use of this test is not yet a reality. Many researchers expect it to expand well beyond the reference laboratory and replace the MMA assay. Antibodies to Intrinsic Factor and Parietal Cells Because intrinsic factor has 2 binding sites, antibodies to intrinsic factor can be binding antibodies that bind to the IF binding site for vitamin, or blocking antibodies that bind to IF at the binding site for the IF receptor on the surface of ileal cells. 1 These are designated anti-if type I and type II antibodies, respectively. It is the type I antibodies that are usually measured. 4 In addition, antibodies may form against parietal cells (anti-pc). The antigen against which these auto-antibodies are produced is the gastric proton pump. 1 Only some pernicious anemia patients have anti-if antibodies, but practically all those who have these anti-if antibodies have pernicious anemia. 9 With the anti-parietal cell antibody test, the situation is reversed. It will reveal most cases of pernicious anemia, but only some of the positives are truly pernicious anemia. Outside of the elderly, white population these statistics change. Anti-IF antibodies are more commonly seen in black and Latin American patients than white patients, 68 whereas anti-pc antibodies are more commonly seen in older individuals than the young. Because of the higher specificity of anti-if for pernicious anemia, it is preferred over anti-pc in diagnosis of suspected pernicious anemia. These antibodies are not detected in food-cobalamin malabsorption. Serum Gastrin Hypochlorhydria increases serum gastrin to re-stimulate acid production by the stomach. This feedback hypergastrinemia is associated with hyperplasia of G cells in the antrum of the stomach. 9 A high fasting serum gastrin level hints at pernicious anemia but can have other causes including other types of gastritis. PA can be virtually confirmed by also testing positive for anti-intrinsic factor antibodies. 9 The gastrin test alone is only somewhat sensitive. High levels are seen in 80% to 90% of patients with pernicious anemia 9,69,70 and some patients with food-cobalamin malabsorption. Gastrin should not be used as an initial test for vitamin deficiency but rather as a confirmatory test. Because this is a surrogate test for vitamin malabsorption and often fails to exceed the gastrin reference range, its use is precarious for making clinical diagnoses. 25 Gastrin levels were shown to be higher in women than men and higher in white and black patients than Latin Americans. 68 Such differences should be taken into account when evaluating test results against reference values. Serum Pepsinogen Like hypergastrinemia, low pepsinogen I or ratio of pepsinogen I to pepsinogen II are fairly sensitive markers of severe food-cobalamin malabsorption, gastritis, and pernicious anemia. 25,70 Pepsinogen II can rise with H. pylori infection that causes severe food-cobalamin malabsorption, leading to a low pepsinogen I/pepsinogen II ratio, but pepsinogen II alone is not independently a significant factor. 25 Pepsinogen, a precursor of pepsin, is secreted by gastric glands in response to stomach acidity. 20 Hence, lack of stomach acidity as a result of gastritis and pernicious anemia will lower the pepsinogen level. As atrophic gastritis progresses, serum pepsinogen I will drop accordingly. 71 Non-Radioactive Oral Vitamin Test The non-radioactive oral vitamin test is a simple, reliable absorption test. 72 Serum cobalamin is measured before and after short-term treatment with oral vitamin. Absorption of vitamin is determined by how much the level increases with treatment. If not sufficiently increased, this is repeated with intrinsic factor added to the oral dose. Greater vitamin level increase with intrinsic factor added indicates lack of intrinsic factor while no further increase indicates another problem such as food-cobalamin malabsorption or intestinal disorder. This test has the unique benefit of testing for the cause of the deficiency without radioactive substances. Summary Newer tests for vitamin deficiency and pernicious anemia allow for higher sensitivity and/or specificity in diagnosing vitamin deficiency and often can be accomplished at lower cost. Test sensitivities and specificities are compiled in Table LABMEDICINE Volume 37 Number 3 March 2006 labmedicine.com Downloaded from

8 Table 4_Summary of Vitamin Deficiency Test Sensitivity and Specificity Test Sensitivity Specificity MCV 17% 73 low Schilling test phase I 61% 72 moderate Serum vitamin % 73, % 50,75 RBC folate low-moderate low-moderate dust high 49 moderate Serum MMA 98% 76 high Urine MMA 79%-86% 77 85%-98% 77,78 Plasma thcy 73-96% 49, % 74 Holo-TC II 100% 79 89% 79 Anti-IF type I 17-73% 70, % 9,74 Anti-PC 52%->90% 9,70 50% 9 Serum gastrin 80-90% 70 50% 70 Serum pepsinogen I/II ratio 82% 70 low 70 Non-radioactive oral absorption test 81% 72 high Note: The Schilling, anti-if, anti-pc, and non-radioactive oral absorption assays test for pernicious anemia, not food-cobalamin malabsorption or other causes of vitamin Therefore, their sensitivities and specificities are listed in regards to detection of pernicious anemia. In all other cases the numbers pertain to detection of vitamin deficiency as a whole. Test sensitivity is particularly important for the diagnosis of subclinical deficiency, which affects millions of elderly individuals, and for atypical presentations of vitamin Note the high sensitivity and specificty of serum MMA and holo-tc II and the lower specificity of plasma thcy. Other factors to take into account when deciding which tests to use include availability, convenience, cost, and whether pernicious anemia or foodcobalamin absorption is suspected. A clinician may require positive results from several tests to confidently diagnose the deficiency or will tailor an assessment regimen to a patient s specific clinical situation. In Figure 4, we suggest the use of serum vitamin then the MMA or IF blocking antibody test followed by the serum gastrin assay if possible. However, many health care centers do not have the facilities or resources to conduct serum MMA measurements. While not ideal, plasma total homocysteine can be used as a screening tool until serum MMA assays are widely available with a more affordable price tag. In the event that a patient is borderline vitamin deficient by a serum vitamin level, the best approach may be a trial therapy with vitamin supplementation before further diagnostic investigation. The authors do not feel that a thorough investigation is always warranted, but when needed the algorithm in Figure 4 should be followed. The holo-tc II test is becoming widely available and may replace several of these follow-up tests. The Schilling test should not be used due to its unreliability. There have been great advances in the treatment of pernicious anemia and related disorders causing vitamin This disease is no longer deadly. The diagnosis is blurred by many choices for laboratory testing, but perhaps the new options, along with a well-devised test regimen, will allow the educated health professional to achieve accurate, early, and cost-effective test results. LM Glossary of Terms AdoCbl adenosylcobalamin CNCbl cyanocobalamin IF intrinsic factor MMA methylmalonic acid MeCbl methylcobalamin OHCbl hydroxocobalamin PA PC TC II thcy THF pernicious anemia parietal cells transcobalamin II total homocysteine tetrahydrofolate 1. Whittingham S, Mackay IR. Autoimmune gastritis: historical antecedents, outstanding discoveries, and unresolved problems. Int Rev Immunol. 2005;24: Schilling RF. Intrinsic factor studies II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B12. J Lab Clin Med. 1953;42: Nobel Prize.org. Updated 16 June The Nobel Foundation. Available at: Accessed 11 Aug Markle HV. Cobalamin. Crit Rev Clin Lab Sci. 1996;33: Micromedex Healthcare Series: Thomson Micromedex, Greenwood Village, Colorado (Edition expires 9/2005). 6. Herrmann W, Geisel J. Vegetarian lifestyle and monitoring of vitamin B12 status. Clin Chim Acta. 2002;326: Martens JH, Barg H, Warren MJ, et al. Microbial production of vitamin B12. Appl Microbiol Biotechnol. 2002;58: Dietary Supplement Fact Sheet: Vitamin B12. Updated 25 May National Institutes of Health Office of Dietary Supplements. Available at: Accessed 11 Aug Andres E, Loukili NH, Noel E, et al. Vitamin B12 (cobalamin) deficiency in elderly patients. CMAJ. 2004;171: Okuda K. Discovery of vitamin B12 in the liver and its absorption factor in the stomach: A historical review. J Gastroenterol Hepatol. 1999;14: Hardeman JG, Limbird LE, Gilman AG, eds. Goodman & Gilman s The Pharmacological Basis of Therapeutics. 10th edition. New York: McGraw Hill; Quadros EV, Nakayama Y, Sequeira JM. The binding properties of the human receptor for the cellular uptake of vitamin B12. Biochem Biophys Res Commun. 2005;327: Lindemans J, Kroes AC, van Geel J, et al. Uptake of transcobalamin II-bound cobalamin by HL-60 cells: effects of differentiation induction. Exp Cell Res. 1989;184: Warren MJ, Raux E, Schubert HL, et al. The biosynthesis of adenosylcobalamin (vitamin B12). Nat Prod Rep. 2002;19: Castle WB. Development of knowledge concerning the gastric intrinsic factor and its relation to pernicious anemia. N Engl J Med. 1953;249: Carmel R. Current concepts in cobalamin Annu Rev Med. 2000;51: Toh BH, van Driel IR, Gleeson PA. Pernicious anemia. N Engl J Med. 1997;337: Karlsson FA, Burman P, Loof L, et al. Major parietal cell antigen in autoimmune gastritis with pernicious anemia is the acid-producing H+,K+-adenosine triphosphatase of the stomach. J Clin Invest. 1988;81: Matthews CK, van Holde KE. Biochemistry. 2nd edition. Menlo Park, NJ: Benjamin/Cummins Publishing Company, Inc; Guyton AC, Hall JE. Textbook of Medical Physiology. 10th edition. Philadelphia, PA: W.B. Saunders Company; Carmel R. Cobalamin, the stomach, and aging. Am J Clin Nutr. 1997;66: Baik HW, Russel RM. Vitamin B12 deficiency in the elderly. Annu Rev Nut. 1999;19: Herbert V. Megaloblastic anemias. Lab Invest. 1985;52: Carmel R. Helicobacter pylori infection and food-cobalamin malabsorption. Dig Dis Sci. 1994;39: Carmel R. Associations of food-cobalamin malabsorption with ethnic origin, age, Helicobacter pylori infections, and serum markers of gastritis. Am J Gastroenterol. 2001;96: Alpers DH. What is new in vitamin B12? Curr Opin Gastroenterol. 2005;21: Teplitsky V, Huminer D, Zoldan J, et al. Hereditary partial transcobalamin II deficiency with neurologic, mental and hematologic abnormalities in children and adults. Isr Med Assoc J. 2003;5: Carmel R. Prevalence of undiagnosed pernicious anemia in the elderly. Am J Clin Nutr. 1996;65: Zittoun J, Zittoun R. Modern clinical testing strategies in cobalamin and folate Semin Hematol. 1999;36: Klee GG. Cobalamin and folate evaluation: measurement of methylmalonic acid and homocysteine vs vitamin B(12) and folate. Clin Chem. 2000;46: Ray JG, Vermeulen MJ, Langman LJ, et al. Persistence of vitamin B12 insufficiency among elderly women after folic acid food fortification. Clin Biochem. 2003;36: Howden CW. Vitamin B12 levels during prolonged treatment with proton pump inhibitors. J Clin Gastroenterol. 2000;30: Desta Z, Zhao X, Shin JG, et al. Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin Pharmacokinet. 2002;41: Sagar M, Janczewska I, Ljungdahl A, et al. Effect of CYP2C19 polymorphism on serum levels of vitamin B12 in patients on long-term omeprazole treatment. Aliment Pharmacol Ther. 1999;13: Downloaded labmedicine.com from March 2006 Volume 37 Number 3 LABMEDICINE 173

9 35. Desouza C, Keebler M, McNamara DB, et al. Drugs affecting homocysteine metabolism: impact on cardiovascular risk. Drugs. 2002;62: Lane LA, Rojas-Fernandez C. Treatment of vitamin b(12)-deficiency anemia: Oral versus parenteral therapy. Ann Pharmacother. 2002;36: Carmel R, Melnyk S, James SJ. Cobalamin deficiency with and without neurologic abnormalities: Differences in homocysteine and methionine metabolism. Blood. 2003;101: Carmel R, Green R, Rosenblatt DS, et al. Update on cobalamin, folate, and homocysteine. Hematology. 2003: Carmel R. Pernicious anemia. The expected findings of very low serum cobalamin levels, anemia, and macrocytosis are often lacking. Arch Intern Med. 1988;148: Ward PC. Modern approaches to the investigation of vitamin B12 Clin Lab Med. 2002;22: Arteaga de Murphy C, Maisterrena J, et al. Rev Invest Clin. 1991;43: Chanarin I, Waters DAW. Failed Schilling tests. Scand J Haemat. 1974;12: Scandurra R, Politi L. Methods of determination of vitamin. Acta Vitaminol Enzymol. 1981;3: Access, Access 2 and UniCel DxI 800 Access Immunoassay Systems. Beckman Coulter. Updated Available at: products/testmenu/access.asp. Accessed 11 Aug Komaromy-Hiller G, Nuttall KL, Ashwood ER. Effect of storage on serum vitamin B12 and folate stability. Ann Clin Lab Sci. 1997;27: Rampersaud GC, Kauwell GP, Bailey LB. Folate: A key to optimizing health and reducing disease risk in the elderly. J Am Coll Nutr. 2003;22: Killmann SA. Effect of deoxyuridine on incorporation of tritiated thymidine: Difference between normoblasts and megaloblasts. Acta Med Scand. 1964;175: Carmel R, Rasmussen K, Jacobsen DW, et al. Comparison of the deoxyuridine suppression test with serum levels of methylmalonic acid and homocysteine in mild cobalamin Br J Haematol. 1996;93: Wickramasinghe SN, Matthews JH. Deoxyuridine suppression: biochemical basis and diagnostic applications. Blood Rev. 1988;2: Lindenbaum J, Savage DG, Stabler SP, et al. Diagnosis of cobalamin deficiency: II. Relative sensitivities of serum cobalamin, methylmalonic acid, and total homocysteine concentrations. Am J Hematol. 1990;34: Kushnir MM, Komaromy-Hiller G, Shushan B, et al. Analysis of dicarboxylic acids by tandem mass spectrometry. High-throughput quantitative measurement of methylmalonic acid in serum, plasma, and urine. Clin Chem. 2001;47: Luo X, Zhang L, Wei H, et al. Methylmalonic acid in amniotic fluid and maternal urine as a marker for neural tube defects. J Huazhong Univ Sci Technolog Med Sci. 2004;24: Refsum H, Smith AD, Ueland PM, et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem. 2004;50: Stabler SP, Allen RH, Savage DG, et al. Clinical spectrum and diagnosis of cobalamin Blood. 1990;76: McMullin MF, Young PB, Bailie KEM, et al. Homocysteine and methylmalonic acid as indicators of folate and vitamin B12 deficiency in pregnancy. Clin Lab Haem. 2001; 23: Hvas AM, Ellegaard J, Nexo E. Increased plasma methylmalonic acid level does not predict clinical manifestations of vitamin B12 Arch Intern Med. 2001;161: Carmel R. Megaloblastic anemias. Curr Opin Hematol. 1994;1: Nelen WL. Hyperhomocysteinaemia and human reproduction. Clin Chem Lab Med. 2001;39: Seshadri S, Beiser A, Selhub J, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer s disease. N Engl J Med. 2002;346: Malouf M, Grimley EJ, Areosa SA. Folic acid with or without vitamin B12 for cognition and dementia. Cochrane Database Syst Rev. 2003;(4):CD Guttormsen AB, Ueland PM, Svarstad E, et al. Kinetic basis of hyperhomocysteinemia in patients with chronic renal failure. Kidney Int. 1997;52: Carmel R. Measuring and interpreting holo-transcobalamin (holo-transcobalamin II). Clin Chem. 2002;48: Ulleland M, Eilertsen I, Quadros EV, et al. Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum. Clin Chem. 2002;48: Nexo E, Christensen AL, Hvas AM. Quantification of holo-transcobalamin, a marker of vitamin B12 Clin Chem. 2002;48: Chen X, Remacha AF, Sarda MP, et al. Influence of cobalamin deficiency compared with that of cobalamin absorption on serum holo-transcobalamin II. Am J Clin Nutr. 2005;81: Hvas AM, Nexo E. Holotranscobalamin a first choice assay for diagnosing early vitamin B12 deficiency? J Intern Med. 2005;257: Loikas S, Lopponen M, et al. RIA for serum holo-transcobalamin: method evaluation in the clinical laboratory and reference interval. Clin Chem. 2003;49: Carmel R. Reassessment of the relative prevalences of antibodies to gastric parietal cell and to intrinsic factor in patients with pernicious anemia: Influence of patient age and race. Clin Exp Immunol. 1992;89: Chanarin I. The megaloblastic anemias. 2nd edition. Oxford, England: Blackwell Scientific Publishers; Carmel R. Pepsinogens and other serum markers in pernicious anemia. Am J Clin Pathol. 1988;90: Yahagi N, Shimizu Y, et al. Corroborative evidence on mass screening method using serum pepsinogen test for long term follow up of gastric cancer patients. Clinical Gastroenterology. 2002;17: Magnus E, Müller C. A new, peroral non-radioactive vitamin B12 absorption test compared with the Schilling test. Eur J Haematol. 1995;54: Oosterhuis WP, Niessen RW, Bossuyt PM, et al. Diagnostic value of the mean corpuscular volume in the detection of vitamin B12 Scand J Clin Lab Invest. 2000;60: Matchar DB, McCrory DC, Millington DS, et al. Performance of the serum cobalamin assay for diagnosis of cobalamin Am J Med Sci. 1994;308: Bolann BJ, Solli JD, Schneede J, et al. Evaluation of indicators of cobalamin deficiency defined as cobalamin-induced reduction in increased serum methylmalonic acid. Clin Chem. 2000;46: Savage DG, Lindenbaum J, Stabler SP, et al. Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am J Med. 1994;96: Kwok T, Cheng G, Lai WK, et al. Use of fasting urinary methylmalonic acid to screen for metabolic vitamin B12 deficiency in older persons. Nutrition. 2004;20: Marin GH, Tentoni J, Cicchetti G. [Megaloblastic anemia: rapid and economical study] Sangre (Barc). 1997;42: Hvas AM, Nexo E. Holotranscobalamin as a predictor of vitamin B12 status. Clin Chem Lab Med. 2003;41: Downloaded 174 from LABMEDICINE Volume Number 3 March 2006 labmedicine.com