Minireviews Open Access
Copyright ©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Diabetes. Jul 15, 2025; 16(7): 107514
Published online Jul 15, 2025. doi: 10.4239/wjd.v16.i7.107514
Metformin-induced vitamin B12 deficiency: An underdiagnosed cause of diabetic neuropathy
Mauricio Alvarez, Natalia Portilla, Oswaldo Rincon, Isaac Guzman, Department of Endocrinology, Hospital Militar Central, Bogotá 110221, Colombia
Andres E Prieto, Internal Medicine Program, University Militar Nueva Granada, Bogotá 110221, Colombia
Diana Moya, Internal Medicine Program, University de la Sabana, Bogotá 110221, Colombia
ORCID number: Mauricio Alvarez (0000-0003-3171-1043).
Author contributions: Alvarez M, Prieto AE, Portilla N, Moya D, Rincon O, and Guzman I contributed to the preparation of the manuscript and approved its final content.
Conflict-of-interest statement: All authors report no relevant conflicts of interest for this article.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Mauricio Alvarez, MD, Department of Endocrinology, Hospital Militar Central, Carrera 7a n 94 a 47, Bogotá 110221, Colombia. mauricioalvarez613@gmail.com
Received: March 25, 2025
Revised: April 24, 2025
Accepted: June 11, 2025
Published online: July 15, 2025
Processing time: 112 Days and 12 Hours

Abstract

Metformin-induced vitamin B12 deficiency is a prevalent condition among patients with type 2 diabetes mellitus. In recent years, a growing body of evidence has demonstrated the association between vitamin B12 deficiency and the onset, progression, and worsening of diabetic neuropathy (DNP) as well as its improvement with supplementation in cases of deficiency. Major clinical guidelines for diabetes and DNP remain vague in their recommendations for B12 measurement and supplementation, and some guidelines do not address it at all. Given that vitamin B12 therapy is an economical, safe, and widely available treatment in most countries and supported by emerging evidence of its potential benefits, greater efforts should be made to promote systematic screening for vitamin B12 deficiency in all patients with DNP before establishing a definitive diagnosis as well as in patients with diabetes with risk factors for deficiency. Vitamin B12 deficiency should be treated in all affected patients, and supplementation should be considered in those with borderline levels when confirmatory diagnostic tests for deficiency are unavailable. Clinical guidelines should place greater emphasis on the recommendations for measuring and supplementing vitamin B12 in these patients.

Key Words: Metformin; Vitamin B12 deficiency; Diabetic neuropathy; Type 2 diabetes mellitus; Clinical guidelines

Core Tip: Metformin-induced vitamin B12 deficiency is common in type 2 diabetes and is increasingly linked to diabetic neuropathy. Despite emerging evidence supporting screening and supplementation, major clinical guidelines provide inconsistent recommendations. Given the safety, affordability, and potential benefits of vitamin B12, systematic screening should be prioritized in patients with diabetes at risk of deficiency, especially those with neuropathy.



INTRODUCTION

Vitamin B12 (cobalamin) deficiency is highly prevalent in diabetes due to multiple factors and is a common cause of neuropathy, megaloblastic anemia, cognitive impairment, and hematological abnormalities, particularly in the elderly population. Its diagnosis is complex and often requires a combination of tests, the interpretation of which demands a deep understanding of physiology. Early diagnosis is crucial, and treatment is typically beneficial for both the clinician and the patient[1].

PHYSIOLOGY OF VITAMIN B12

In healthy adults total body stores of cobalamin range from 3-5 mg, with a recommended daily intake of 2.4 µg obtained from animal-based foods[2]. Vitamin B12 deficiency can result from inadequate dietary intake or malabsorptive disorders. Once a state of deficiency is reached, the vitamin becomes insufficient to support key biochemical reactions requiring it as a cofactor, leading to functional disruption and ultimately impairing cellular integrity. Normal absorption of vitamin B12 begins with its binding to salivary haptocorrin, which transports it to the stomach where intrinsic factor produced by gastric parietal cells is required for further processing. Dietary B12, along with B12 secreted in bile and drained into the duodenum, remains bound to haptocorrin in the upper small intestine. There, trypsin degrades haptocorrin, allowing B12 to bind intrinsic factor, which facilitates its absorption via functional cubam receptors in the terminal ileum. Within intestinal epithelial cells lysosomes degrade intrinsic factor, and B12 is exported into circulation via multidrug resistance protein 1. In plasma, B12 circulates bound to transcobalamin, which delivers it to target cells by binding to CD320 receptors. Once internalized, lysosomes degrade transcobalamin, making B12 available for conversion into adenosylcobalamin in the mitochondria and methylcobalamin in the cytosol, both serving as essential cofactors for B12-dependent reactions. These include the mitochondrial conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A and the cytosolic conversion of homocysteine to methionine (Figure 1)[3].

Figure 1
Figure 1 Folate and methionine cycles. BHMT: Betaine-homocysteine methyltransferase; SAM: S-adenosyl methionine; SAH: S-adenosyl homocysteine.

Vitamin B12 and folates are closely interconnected due to their cooperative roles in metabolism, making their deficiencies clinically indistinguishable. Both lead to impaired DNA synthesis, prolongation of the S phase of the cell cycle, and maturation arrest. Persistent failure in the folate-dependent conversion of deoxyuridine to deoxythymidine results in DNA strand breaks, fragmentation, and apoptosis[4]. These alterations manifest as the megaloblastic changes observed in the bone marrow where basophilic erythroblasts predominate over more mature forms, contributing to progressive anemia.

The resulting symptoms include weakness, palpitations, fatigue, dyspnea, and in severe cases syncope. Progressive pallor combined with hemolysis-induced jaundice due to increased erythrocyte rigidity and abnormal membrane proteins shortens their lifespan by 30%-50%, producing the characteristic lemon-yellow tint[5]. Although all blood cell lines are affected, erythrocytes exhibit the most pronounced changes, with oval macrocytes and prominent anisopoikilocytosis. Megaloblasts become evident in peripheral blood when hematocrit levels drop below 20%. Neutrophils frequently show hypersegmentation, exceeding the usual 2-5 lobes, with 6 or more being characteristic. Platelets typically decrease by more than 10%, and these hematologic abnormalities may persist beyond treatment[6]. The interaction of vitamin B12 with other nutrients is essential for understanding its physiology and manifestations. Coexisting iron deficiency or thalassemia may mask typical megaloblastic changes. Regarding folate metabolism, B12 deficiency disrupts the conversion of methylene tetrahydrofolate to tetrahydrofolate, which is necessary for thymidine synthesis during DNA replication (Figure 1)[1,7].

NEUROPATHIES ASSOCIATED WITH VITAMIN B12 DEFICIENCY

Vitamin B12 deficiency is clinically recognized as a factor in neurological disorders such as dementia, cognitive impairment, and depression. Additionally, it contributes to demyelination in the peripheral and central nervous systems and has been linked to peripheral neuropathy, sensory loss, and weakness in the lower limbs, particularly in older adults. Specifically, B12 deficiency is associated with long-fiber neuropathy (type A), affecting both sensory and motor functions[8-10]. Neurological manifestations of B12 deficiency are heterogeneous. A retrospective review of 32 patients with neurological abnormalities due to B12 deficiency who underwent nerve conduction studies revealed that the most common symptoms were paresthesia, hyporeflexia, ataxia, and weakness in the majority (62%). Electromyography showed that 50.00% had axonal damage, 9.30% had demyelinating neuropathy, and 18.75% exhibited mixed characteristics. Sensory impairment alone was observed in 34.30%, while 43.75% presented a mixed sensory-motor pattern[11]. The exact mechanism underlying neurological complications remains undetermined. It is hypothesized that reduced succinyl-coenzyme A synthesis and secondary accumulation of methylmalonic acid (MMA) may contribute to the pathological process by promoting the formation of odd-chain and branched-chain fatty acids. Additionally, these metabolic changes may be linked to an inflammatory state with oxidative stress and microvascular disease associated with hyperhomocysteinemia[12,13].

DIAGNOSIS OF VITAMIN B12 DEFICIENCY

Clinical suspicion is paramount for diagnosing deficiency, especially in cases with a high pretest probability of severe or non-severe B12 deficiency. This distinction is based on the fact that severe deficiency is more strongly associated with megaloblastic anemia and overt neurological complications than milder deficiencies, which may result from a vegan diet, medication use (e.g., metformin, proton pump inhibitors, histamine receptor antagonists), or non-autoimmune gastritis[2]. Laboratory findings typically include the hematological abnormalities described above, along with increased bilirubin and lactate dehydrogenase (LDH), with LDH-1 predominating over LDH-2. Additionally, there is a characteristic elevation in serum iron and ferritin due to impaired iron utilization[6].

Serum B12 levels, commonly used as an initial screening test, have limited sensitivity and specificity when interpreted in isolation. Low levels do not always indicate deficiency, and normal levels do not necessarily exclude it. Studies define low B12 status as levels < 148 pmol/L, while marginal or relative deficiency is characterized by values between 148-221 pmol/L, and even some consider values up to 260 pmol/L[14]. Approximately 70%-90% of circulating B12 is bound to haptocorrin and is unavailable for cellular use, whereas only 10%-30% is bound to transcobalamin, its functional transport protein[1,6,14]. Based on this distribution, measuring the fraction of B12 bound to transcobalamin [holotranscobalamin (holoTC) or active B12] provides a more accurate assessment of functional B12 status and absorptive capacity. Based on this distribution measuring the fraction of B12 bound to transcobalamin, known as holoTC or active B12, offers a more accurate assessment of functional B12 status and absorptive capacity as demonstrated in previous studies[15].

Total serum B12 concentrations can be nonspecifically influenced by conditions that alter haptocorrin expression, such as malignancy, pregnancy, liver disease, and autoimmune disorders as well as by analytical interference from circulating antibodies. In contrast holoTC levels are primarily affected by transcobalamin gene polymorphisms (e.g., total transcobalamin concentrations are approximately 20% lower in individuals with the 776GG genotype compared with those with the 776CC genotype, with intermediate levels observed in individuals with the 776GC genotype) as well as by analytical interference and recent vitamin B12 intake. Despite its higher cost and limited availability, holoTC is currently considered the earliest and most specific marker of B12 deficiency[15].

The measurement of B12-dependent reaction substrates, such as MMA and homocysteine, serves as a sensitive indicator of deficiency. Elevated levels are detected in over 90% of patients with B12 deficiency, even before serum B12 levels fall below the normal range, making them highly sensitive diagnostic tools. MMA can be measured in plasma or serum, whereas homocysteine is best assessed in plasma. Between them MMA is more specific for B12 deficiency and remains elevated even after treatment initiation[2,14]. MMA increases exclusively in B12 deficiency, whereas homocysteine levels also rise in folate and pyridoxine deficiencies as well as in hypothyroidism[14].

PATHOPHYSIOLOGY OF DIABETIC NEUROPATHY

Diabetic neuropathy (DNP) is a heterogeneous group of conditions with widely varying pathology that remains incompletely understood and multifactorial in origin. However, multiple pathophysiological mechanisms contribute to its development, including metabolic disturbances such as hyperglycemia, hypoinsulinemia, growth factor abnormalities, and dyslipidemia. These alterations activate various pathophysiological pathways, ultimately leading to oxidative stress and nerve dysfunction[16,17]. One key mechanism involves the increased affinity of aldose reductase for glucose. Aldose reductase catalyzes the conversion of glucose to sorbitol, resulting in sorbitol accumulation and a reduction in myo-inositol within nerve tissue, a process known as the polyol pathway. This leads to osmotic stress, impairing Schwann cell function and contributing to nerve fiber degeneration. Additionally, nicotinamide adenine dinucleotide phosphate depletion reduces the capacity of the cell to neutralize reactive oxygen species, further exacerbating oxidative damage. Furthermore, free radicals, oxidants, and certain unidentified metabolic factors activate the nuclear enzyme poly(ADP-ribose) polymerase, which has been implicated in the progression of DNP[16].

An increase in oxidative stress has been demonstrated in various studies, with elevated levels of oxidative stress markers such as superoxide ions and peroxynitrite observed in patients with hyperglycemia. This is accompanied by a reduction in antioxidant substances in individuals with DNP[18]. Another key factor is the disruption of Na+/K+ adenosine triphosphatase pump activity, attributed to alterations in the polyol pathway and inhibition of myo-inositol absorption. These changes impair nerve cell depolarization, ultimately leading to slowed nerve conduction[16]. Additionally, hyperglycemia induces endoplasmic reticulum (ER) stress, leading to the accumulation of unfolded or misfolded proteins within the ER lumen. This activates the unfolded protein response, a signaling cascade aimed at restoring normal ER function. However, during prolonged or extreme stress, the unfolded protein response may become overwhelmed, triggering apoptotic pathways[19].

Another key component in the pathophysiology of DNP is microvascular insufficiency, which represents the second major group of pathophysiological mechanisms. This involves the activation of protein kinase C, driven by both hyperglycemia and altered fatty acid metabolism, leading to increased production of vasoconstrictive, angiogenic, and chemotactic cytokines such as transforming growth factor-β, vascular endothelial growth factor, endothelin, and intercellular adhesion molecules. These factors contribute to the loss of the vasa nervorum, the small blood vessels that supply peripheral nerves[16,18]. Furthermore, growing evidence suggests a deficiency of nerve growth factor and neuropeptides such as substance P and calcitonin gene-related peptide that further contribute to microvascular insufficiency and ischemic nerve damage. In addition reduced nitric oxide levels and increased reactive oxygen species activity exacerbate hypoxia and microvascular injury[16]. Finally, the presence of autoantibodies targeting neuronal cell epitopes has been reported. For instance a 12% incidence of a motor neuropathy variant has been observed in patients with diabetes and is associated with anti-GM1 monoclonal ganglioside autoantibodies. Additionally, individuals with diabetes have an 11-fold increased risk of developing chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, vasculitis, and monoclonal gammopathies[18].

DIAGNOSIS OF DNP

Clinical suspicion of DNP arises based on symptoms, which vary depending on the type of neuropathy suspected and the organ(s) involved. The American Diabetes Association recommends screening for peripheral and autonomic neuropathy in individuals with type 1 diabetes of more than a 5-year duration and annually in all individuals with type 2 diabetes, using medical history and simple diagnostic tests[18]. For peripheral neuropathy early symptoms such as pain and dysesthesia (unpleasant burning and tingling sensations) suggest small fiber involvement, whereas numbness and loss of protective sensation indicate large fiber involvement[18]. Initial screening for small fiber function includes assessing pinprick and temperature sensation. Large fiber function should be evaluated by testing lower extremity reflexes, vibration perception, and light touch sensation using a 10 g monofilament on the dorsum of the great toe as well as assessing bilateral ankle reflexes and proprioception[18,20]. At least one test for small fiber function and one for large fiber function is recommended[20]. Additionally, symptoms and clinical signs indicative of autonomic neuropathy should be assessed.

Key manifestations include resting tachycardia, orthostatic hypotension, gastroparesis, constipation, diarrhea, fecal incontinence, erectile dysfunction, neurogenic bladder, and sudomotor dysfunction, which may present as either increased or decreased sweating. Further diagnostic testing may be considered depending on the affected organ system and may include cardiovascular autonomic testing, sweat testing, urodynamic studies, gastric emptying studies, or endoscopic procedures such as colonoscopy[18]. Since DNP is a diagnosis of exclusion, other potential causes of neuropathy should be systematically evaluated once it is suspected. These include exposure to toxins (e.g., alcohol), neurotoxic medications (e.g., chemotherapy), vitamin B12 deficiency, hypothyroidism, renal disease, malignancies (e.g., multiple myeloma, bronchogenic carcinoma), infections (e.g., human immunodeficiency virus), chronic inflammatory demyelinating neuropathy, inherited neuropathies, and vasculitis[18].

In cases with atypical clinical features, electrodiagnostic studies and/or referral to a neurologist are recommended. Atypical clinical features in DNP may include asymmetric or focal neuropathic symptoms, rapid progression of symptoms, or the presence of unusual sensory disturbances such as severe pain, hyperalgesia, or allodynia that are disproportionate to the extent of nerve damage. Additionally, if there are early-onset symptoms, significant weakness, or evidence of other systemic manifestations (e.g., autonomic dysfunction, muscle atrophy, or cranial nerve involvement), it may suggest an alternative diagnosis, such as an inflammatory, autoimmune neuropathy or neuropathy secondary to another underlying systemic condition[20]. Clinical instruments, such as questionnaires that assess signs and symptoms or combine both, have been used for neuropathy evaluation. Examples include the neurological impairment score, which assesses sensory and motor function, and the neurological symptom score[21]. It is important to note that the gold standard for assessing intraepidermal nerve fiber density is a skin punch biopsy, though its use is limited due to its invasive nature and restricted availability[20].

Since the 2009 Toronto consensus, neurology societies have defined confirmed diabetic sensorimotor polyneuropathy (DSP) as requiring both abnormal nerve conduction studies and the presence of neuropathic symptoms or signs. Probable DSP is diagnosed when both symptoms and signs are present, whereas possible DSP requires either symptoms or signs alone[22]. The most challenging diagnosis involves small fiber neuropathy as electrophysiological studies may appear normal. In such cases diagnosis may rely on specialized assessments of small fiber function, including sudomotor testing or skin biopsy with measurement of intraepidermal nerve fiber density[22].

CROSSROADS BETWEEN DNP AND VITAMIN B12 DEFICIENCY-RELATED NEUROPATHY

DNP and neuropathy due to vitamin B12 deficiency share several clinical and pathophysiological features, which can lead to diagnostic challenges in clinical practice. Both conditions primarily affect the peripheral nervous system and can present with sensory loss, paresthesia, weakness, and hyporeflexia, particularly in the lower limbs. Additionally, both neuropathies can manifest as mixed sensory-motor involvement and may exhibit axonal damage, with potential demyelinating features. This overlap is particularly relevant in older adults in whom vitamin B12 deficiency is more prevalent and diabetes is a common comorbidity. Given that nerve conduction studies may show similar patterns in both conditions, distinguishing between them requires careful clinical assessment, including a detailed history, biochemical evaluation of B12 levels, and consideration of risk factors. Failure to identify and treat B12 deficiency in diabetic patients with neuropathy may result in persistent or worsening neurological symptoms despite standard DNP management, underscoring the importance of routine screening and targeted intervention in this population.

ASSOCIATION BETWEEN VITAMIN B12 DEFICIENCY AND DNP

DNP is a well-known and extensively studied complication of diabetes, affecting approximately 50% of individuals at some point in their lives[23-26]. For several decades, research has aimed to document the relationship between DNP and vitamin B12 deficiency, which is often secondary to metformin use in this patient population. This is particularly relevant given that approximately 120 million patients are prescribed metformin[27-31]. The association between DNP and vitamin B12 deficiency has been identified in multiple studies, linking this deficiency to the onset, progression, and persistence of DNP[32-35].

Vitamin B12 deficiency is a known adverse effect of metformin use, and various studies have attempted to determine its prevalence in patients with diabetes. However, the reported prevalence varies widely, ranging from 5% to 40%[25] as it is influenced by factors such as the studied population, the diagnostic cutoff points for deficiency, age groups, study characteristics, and the dose and duration of metformin use[24]. Studies have investigated the mechanisms by which metformin causes vitamin B12 deficiency, which is believed to result from various factors, including altered intestinal motility, bacterial overgrowth, and impaired absorption of the vitamin. Once deficiency occurs, it is characterized by neurological manifestations that can be mistaken for DNP[25]. Various studies have sought to determine the factors associated with metformin use and the development of neuropathy, considering aspects such as the duration of metformin treatment before disease onset, vitamin B12 levels, age, metformin dose, among others.

In 2016 an observational study conducted in South Africa analyzed data from patients who had been taking metformin for at least 6 months without baseline cyanocobalamin supplementation and without other risk factors for deficiency, such as alcoholism, gastric surgeries, malabsorption syndromes, or infections[24]. Vitamin B12 deficiency was defined as levels < 150 pmol/L. A total of 121 participants were included, and the prevalence of vitamin B12 deficiency among patients with type 2 diabetes taking metformin was found to be 28.1%. However, no association between vitamin B12 deficiency and peripheral neuropathy was observed[24]. This contrasts with a study conducted in 2019 in Colombia at a tertiary-level hospital where a cross-sectional study was performed to estimate the prevalence of DNP in patients with vitamin B12 deficiency as well as those with low and normal vitamin B12 levels. Vitamin B12 levels were classified as follows: Deficiency (< 200 pg/mL); gray zone (200-300 pg/mL); and normal (> 300 pg/mL). As in the previous study, the Michigan Neuropathy Screening Instrument was used to diagnose DNP. The mean metformin dose was 1536 mg, and the mean duration of use was 108 months. Altered vitamin B12 levels were observed in 29% of patients. Metformin dose was significantly associated with vitamin B12 levels, with higher doses linked to lower vitamin B12 levels. Notably, a significant relationship was observed between the diagnosis of DNP and low vitamin B12 levels [coefficient: -116.9; 95% confidence interval (CI): -165.8 to -68.0]. Additionally, 64% of patients with vitamin B12 levels in the gray zone also had DNP[25].

A more recent meta-analysis published in 2022 evaluated studies conducted between 2010 and 2022, including 17 studies: 13 cross-sectional; 3 cohort; and 1 case-control. The prevalence of vitamin B12 deficiency was significantly higher in patients receiving metformin (23.16%) compared with those not receiving metformin (17.4%) (odd ratios = 2.95, 95%CI: 2.18-4.00, P = 0.001). The meta-analysis also found no association between proton pump inhibitor use and vitamin B12 deficiency. However, the incidence of deficiency increased after 4 years of metformin use, and higher doses were associated with a greater risk of deficiency[22].

Furthermore, another meta-analysis published in 2022 found that vitamin B12 supplementation reduced neuropathy symptoms and pain scores compared with no treatment; however, the studies exhibited high heterogeneity[36]. Recently, another study found that treatment with a combination of 10 elements (palmitoylethanolamide, superoxide dismutase, alpha-lipoic acid, vitamins B12, B1, B6, and E, magnesium, zinc, and nicotinamide) for 6 months at a daily dose of two tablets significantly improved pain, vibration perception threshold, and vitamin B12 levels in individuals with DNP[37]. These findings support the conclusion that vitamin B12 deficiency is associated with an increased risk of neuropathy, and it is likely that vitamin B12 supplementation benefits multiple outcomes in patients with DNP.

WHAT DO THE GUIDELINES SAY?

Currently, vitamin B12 levels are routinely monitored to assess deficiency and if necessary initiate supplementation. Among the guidelines addressing vitamin B12 deficiency, the 2024 National Institute for Health and Care Excellence guidelines provide general recommendations on whom to screen. Screening is advised for individuals presenting with at least one symptom of deficiency and at least one risk factor for developing it, such as metformin use[38]. However, the guidelines do not specify screening frequency (Table 1). Another key document is the Standards of Care in Diabetes-2025 guideline. While previous editions had already described the association between metformin use and vitamin B12 deficiency, the 2025 edition emphasizes its importance, particularly in patients with anemia and those diagnosed with DNP. Regarding metformin use, the guideline highlights an increased risk of deficiency in patients taking doses exceeding 1500 mg/day or those who have been on the drug for more than 4-5 years. Periodic monitoring is recommended although no specific interval is established[39].

Table 1 Clinical guideline recommendations on metformin-induced vitamin B12 deficiency.
Guideline
Recommendation
Monitoring frequency
Treatment
Ref.
British Committee for Standards in HaematologyDoes not recommend routine screening, but suggests evaluation if clinical suspicion exists; highlights the importance of ruling out pernicious anemiaNot specifiedIf due to metformin: Oral cobalamin 50 μg/day for 1 month; prophylactic supplementation not recommended[46]
American Association of Clinical Endocrinologists and American College of Endocrinology-Clinical Practice Guidelines-2015Mentions the risk of deficiency in metformin usersNot specifiedNo treatment specified[40]
Diabetes CanadaDoes not mention vitamin B12 deficiencyNot mentionedNot mentioned[42]
Diabetes Research and Clinical Practice-2023Mentions vitamin B12 only as a differential diagnosisNot mentionedNot mentioned
Vitamin B12 deficiency in over 16 seconds: Diagnosis and management, guidance NICERecommend screening in patients with at least one symptom and one risk factor (such as metformin use)Not specifiedOral or IM vitamin B12; reevaluate after 3 months; consider discontinuing metformin if it is the cause[38]
ADA Standards of Care in Diabetes-2025Acknowledges the risk of deficiency in patients with neuropathy or anemia, especially those taking > 1500 mg/day or using it for > 4-5 yearsPeriodic monitoring recommended, no specific interval definedNo specific treatment recommended[39]

At this time there are no other guidelines that establish recommendations, although in 2016 the American Association of Clinical Endocrinologists and American College of Endocrinology also mentioned this in its guideline[40]. More specific recommendations need to be established in order to adequately follow patients. Some guidelines for the management of DNP do not mention the importance of vitamin B12 deficiency, while others refer to it only as a differential diagnosis rather than a potential initiator, perpetuator, or exacerbating factor of DNP. Additionally, they do not emphasize the need to address this deficiency before initiating treatment or at least as an adjunct to the management of DNP[41-43].

TREATMENT AND SUPPLEMENTATION

When it comes to treating vitamin B12 deficiency, the 2024 National Institute for Health and Care Excellence guidelines provide a broad framework covering both diagnosis and management. While they do not offer case-specific recommendations, they do highlight important considerations, particularly for deficiency linked to metformin use. According to the guidelines, vitamin B12 can be administered either intramuscularly or orally, with the choice depending on patient preference and clinical judgment. They also emphasize reassessing the need for continued metformin use. A standard daily dose of 1 mg of vitamin B12 is recommended, with a follow-up evaluation scheduled 3 months after starting supplementation or sooner if clinically necessary[38].

However, when it comes to supplementation specifically for DNP, no formal guidelines currently exist. While some studies have failed to show a clear benefit in improving peripheral nerve function, others have reported promising results. One randomized controlled trial found that taking 1 mg of vitamin B12 daily for 12 months led to improvements in neurophysiological parameters, motor function, pain scores, and quality of life[44]. Additionally, research comparing intramuscular and oral administration suggests that both routes provide comparable therapeutic effects[45].

The Hematology Society guidelines define vitamin B12 deficiency as levels below 200 ng/L (or 148 pmol/L, depending on the assay). Although routine screening is not specifically recommended for patients with diabetes on metformin, testing should be considered if a deficiency is suspected. The guidelines also highlight the importance of screening for pernicious anemia in these patients as those diagnosed require lifelong supplementation. If pernicious anemia is ruled out and the deficiency is attributed to metformin use, treatment with 50 μg of oral cobalamin per day for 1 month is advised. Interestingly, prophylactic supplementation is not explicitly recommended for these patients[46].

CONCLUSION

Vitamin B12 deficiency is highly prevalent among patients with diabetes mellitus, particularly those with DNP, but diagnosing this deficiency remains challenging due to the lack of a universal diagnostic threshold, limited availability of confirmatory tests, and frequent underdiagnosis in patients with borderline levels. Furthermore, the variability in treatment thresholds across studies complicates clinical decision-making and the development of guideline recommendations. Recent evidence highlights a direct association between vitamin B12 deficiency and the progression of DNP, with supplementation showing potential benefits in improving various outcomes in patients with low or borderline levels of vitamin B12. Despite some guidelines advocating for increased screening, many recommendations remain unclear, and some fail to address the issue altogether. Given that vitamin B12 therapy is an economical, safe, and widely accessible treatment, there is an urgent need for systematic screening for vitamin B12 deficiency in all patients with DNP and those at risk. All affected patients should receive treatment, and supplementation should be considered for those with borderline levels, particularly when confirmatory diagnostic tests are unavailable. Clinical guidelines must place greater emphasis on the measurement and supplementation of vitamin B12 in this population.

Footnotes

Provenance and peer review: Invited article; Externally peer reviewed.

Peer-review model: Single blind

Specialty type: Endocrinology and metabolism

Country of origin: Colombia

Peer-review report’s classification

Scientific Quality: Grade A, Grade A, Grade D, Grade D

Novelty: Grade A, Grade A, Grade D, Grade D

Creativity or Innovation: Grade A, Grade B, Grade D, Grade D

Scientific Significance: Grade A, Grade B, Grade C, Grade D

P-Reviewer: Bharara T; Ulasoglu C S-Editor: Wu S L-Editor: Filipodia P-Editor: Xu ZH

References
1.  Green R. Vitamin B(12) deficiency from the perspective of a practicing hematologist. Blood. 2017;129:2603-2611.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 139]  [Cited by in RCA: 197]  [Article Influence: 24.6]  [Reference Citation Analysis (0)]
2.  Obeid R, Andrès E, Češka R, Hooshmand B, Guéant-Rodriguez RM, Prada GI, Sławek J, Traykov L, Ta Van B, Várkonyi T, Reiners K;  The Vitamin B Consensus Panelists Group. Diagnosis, Treatment and Long-Term Management of Vitamin B12 Deficiency in Adults: A Delphi Expert Consensus. J Clin Med. 2024;13:2176.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in RCA: 9]  [Reference Citation Analysis (0)]
3.  Nielsen MJ, Rasmussen MR, Andersen CB, Nexø E, Moestrup SK. Vitamin B12 transport from food to the body's cells--a sophisticated, multistep pathway. Nat Rev Gastroenterol Hepatol. 2012;9:345-354.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 242]  [Cited by in RCA: 263]  [Article Influence: 20.2]  [Reference Citation Analysis (0)]
4.  Koury MJ, Horne DW, Brown ZA, Pietenpol JA, Blount BC, Ames BN, Hard R, Koury ST. Apoptosis of late-stage erythroblasts in megaloblastic anemia: association with DNA damage and macrocyte production. Blood. 1997;89:4617-4623.  [PubMed]  [DOI]
5.  Coller BS. Blood at 70: its roots in the history of hematology and its birth. Blood. 2015;126:2548-2560.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 21]  [Cited by in RCA: 25]  [Article Influence: 2.5]  [Reference Citation Analysis (0)]
6.  Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013;368:149-160.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 732]  [Cited by in RCA: 746]  [Article Influence: 62.2]  [Reference Citation Analysis (0)]
7.  Green R, Kuhl W, Jacobson R, Johnson C, Carmel R, Beutler E. Masking of macrocytosis by alpha-thalassemia in blacks with pernicious anemia. N Engl J Med. 1982;307:1322-1325.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 37]  [Cited by in RCA: 33]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
8.  Leishear K, Boudreau RM, Studenski SA, Ferrucci L, Rosano C, de Rekeneire N, Houston DK, Kritchevsky SB, Schwartz AV, Vinik AI, Hogervorst E, Yaffe K, Harris TB, Newman AB, Strotmeyer ES; Health, Aging and Body Composition Study. Relationship between vitamin B12 and sensory and motor peripheral nerve function in older adults. J Am Geriatr Soc. 2012;60:1057-1063.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 70]  [Cited by in RCA: 64]  [Article Influence: 4.9]  [Reference Citation Analysis (0)]
9.  Saperstein DS, Barohn RJ. Peripheral Neuropathy Due to Cobalamin Deficiency. Curr Treat Options Neurol. 2002;4:197-201.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 40]  [Cited by in RCA: 28]  [Article Influence: 1.2]  [Reference Citation Analysis (0)]
10.  Ekabe CJ, Kehbila J, Abanda MH, Kadia BM, Sama CB, Monekosso GL. Vitamin B12 deficiency neuropathy; a rare diagnosis in young adults: a case report. BMC Res Notes. 2017;10:72.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 7]  [Cited by in RCA: 11]  [Article Influence: 1.4]  [Reference Citation Analysis (0)]
11.  Bouattour N, Sakka S, Farhat N, Kacem HH, Hdiji O, Dammak M, Mhiri C. Vitamin B12 deficiency neuropathy: A clinical and electrophysiological study. Neurophysiol Clin. 2018;48:130.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 2]  [Cited by in RCA: 5]  [Article Influence: 0.7]  [Reference Citation Analysis (0)]
12.  Green R, Kinsella LJ. Current concepts in the diagnosis of cobalamin deficiency. Neurology. 1995;45:1435-1440.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 155]  [Cited by in RCA: 125]  [Article Influence: 4.2]  [Reference Citation Analysis (0)]
13.  Scalabrino G, Carpo M, Bamonti F, Pizzinelli S, D'Avino C, Bresolin N, Meucci G, Martinelli V, Comi GC, Peracchi M. High tumor necrosis factor-alpha [corrected] levels in cerebrospinal fluid of cobalamin-deficient patients. Ann Neurol. 2004;56:886-890.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 40]  [Cited by in RCA: 41]  [Article Influence: 2.0]  [Reference Citation Analysis (0)]
14.  Green R, Allen LH, Bjørke-Monsen AL, Brito A, Guéant JL, Miller JW, Molloy AM, Nexo E, Stabler S, Toh BH, Ueland PM, Yajnik C. Vitamin B(12) deficiency. Nat Rev Dis Primers. 2017;3:17040.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 373]  [Cited by in RCA: 538]  [Article Influence: 67.3]  [Reference Citation Analysis (0)]
15.  Bor MV, Nexø E, Hvas AM. Holo-transcobalamin concentration and transcobalamin saturation reflect recent vitamin B12 absorption better than does serum vitamin B12. Clin Chem. 2004;50:1043-1049.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 49]  [Cited by in RCA: 47]  [Article Influence: 2.2]  [Reference Citation Analysis (0)]
16.  Pathak R, Sachan N, Chandra P. Mechanistic approach towards diabetic neuropathy screening techniques and future challenges: A review. Biomed Pharmacother. 2022;150:113025.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 2]  [Cited by in RCA: 23]  [Article Influence: 7.7]  [Reference Citation Analysis (0)]
17.  Dewanjee S, Das S, Das AK, Bhattacharjee N, Dihingia A, Dua TK, Kalita J, Manna P. Molecular mechanism of diabetic neuropathy and its pharmacotherapeutic targets. Eur J Pharmacol. 2018;833:472-523.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 102]  [Cited by in RCA: 180]  [Article Influence: 25.7]  [Reference Citation Analysis (0)]
18.  American Diabetes Association Professional Practice Committee. 12. Retinopathy, Neuropathy, and Foot Care: Standards of Care in Diabetes-2024. Diabetes Care. 2024;47:S231-S243.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 41]  [Cited by in RCA: 74]  [Article Influence: 74.0]  [Reference Citation Analysis (0)]
19.  Albers JW, Pop-Busui R. Diabetic neuropathy: mechanisms, emerging treatments, and subtypes. Curr Neurol Neurosci Rep. 2014;14:473.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 209]  [Cited by in RCA: 205]  [Article Influence: 18.6]  [Reference Citation Analysis (0)]
20.  Dillon BR, Ang L, Pop-Busui R. Spectrum of Diabetic Neuropathy: New Insights in Diagnosis and Treatment. Annu Rev Med. 2024;75:293-306.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 13]  [Cited by in RCA: 19]  [Article Influence: 19.0]  [Reference Citation Analysis (0)]
21.  Vinik AI, Nevoret ML, Casellini C, Parson H. Diabetic neuropathy. Endocrinol Metab Clin North Am. 2013;42:747-787.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 186]  [Cited by in RCA: 221]  [Article Influence: 18.4]  [Reference Citation Analysis (0)]
22.  Smith AG, Singleton JR. Diabetic neuropathy. Continuum (Minneap Minn). 2012;18:60-84.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 23]  [Cited by in RCA: 37]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
23.  Kakarlapudi Y, Kondabolu SK, Tehseen Z, Khemani V, J SK, Nousherwani MD, Saleem F, Abdelhameed AN. Effect of Metformin on Vitamin B12 Deficiency in Patients With Type 2 Diabetes Mellitus and Factors Associated With It: A Meta-Analysis. Cureus. 2022;14:e32277.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in RCA: 4]  [Reference Citation Analysis (0)]
24.  Ahmed MA, Muntingh G, Rheeder P. Vitamin B12 deficiency in metformin-treated type-2 diabetes patients, prevalence and association with peripheral neuropathy. BMC Pharmacol Toxicol. 2016;17:44.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 30]  [Cited by in RCA: 48]  [Article Influence: 5.3]  [Reference Citation Analysis (0)]
25.  Alvarez M, Sierra OR, Saavedra G, Moreno S. Vitamin B12 deficiency and diabetic neuropathy in patients taking metformin: a cross-sectional study. Endocr Connect. 2019;8:1324-1329.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 45]  [Cited by in RCA: 35]  [Article Influence: 5.8]  [Reference Citation Analysis (0)]
26.  Jayabalan B, Low LL. Vitamin B supplementation for diabetic peripheral neuropathy. Singapore Med J. 2016;57:55-59.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 38]  [Cited by in RCA: 41]  [Article Influence: 5.1]  [Reference Citation Analysis (0)]
27.  Hansen CS, Jensen JS, Ridderstråle M, Vistisen D, Jørgensen ME, Fleischer J. Vitamin B12 deficiency is associated with cardiovascular autonomic neuropathy in patients with type 2 diabetes. J Diabetes Complications. 2017;31:202-208.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 12]  [Cited by in RCA: 20]  [Article Influence: 2.5]  [Reference Citation Analysis (0)]
28.  Farvid MS, Homayouni F, Amiri Z, Adelmanesh F. Improving neuropathy scores in type 2 diabetic patients using micronutrients supplementation. Diabetes Res Clin Pract. 2011;93:86-94.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 49]  [Cited by in RCA: 56]  [Article Influence: 4.0]  [Reference Citation Analysis (0)]
29.  Li S, Chen X, Li Q, Du J, Liu Z, Peng Y, Xu M, Li Q, Lei M, Wang C, Zheng S, Zhang X, Yu H, Shi J, Tao S, Feng P, Tian H. Effects of acetyl-L-carnitine and methylcobalamin for diabetic peripheral neuropathy: A multicenter, randomized, double-blind, controlled trial. J Diabetes Investig. 2016;7:777-785.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 37]  [Cited by in RCA: 39]  [Article Influence: 4.3]  [Reference Citation Analysis (0)]
30.  Stracke H, Lindemann A, Federlin K. A benfotiamine-vitamin B combination in treatment of diabetic polyneuropathy. Exp Clin Endocrinol Diabetes. 1996;104:311-316.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 100]  [Cited by in RCA: 82]  [Article Influence: 5.1]  [Reference Citation Analysis (1)]
31.  Bell DS. Metformin-induced vitamin B12 deficiency presenting as a peripheral neuropathy. South Med J. 2010;103:265-267.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 67]  [Cited by in RCA: 70]  [Article Influence: 4.7]  [Reference Citation Analysis (0)]
32.  Yang W, Cai X, Wu H, Ji L. Associations between metformin use and vitamin B(12) levels, anemia, and neuropathy in patients with diabetes: a meta-analysis. J Diabetes. 2019;11:729-743.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 36]  [Cited by in RCA: 61]  [Article Influence: 10.2]  [Reference Citation Analysis (0)]
33.  Gupta K, Jain A, Rohatgi A. An observational study of vitamin b12 levels and peripheral neuropathy profile in patients of diabetes mellitus on metformin therapy. Diabetes Metab Syndr. 2018;12:51-58.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 25]  [Cited by in RCA: 29]  [Article Influence: 4.1]  [Reference Citation Analysis (0)]
34.  Alharbi TJ, Tourkmani AM, Abdelhay O, Alkhashan HI, Al-Asmari AK, Bin Rsheed AM, Abuhaimed SN, Mohammed N, AlRasheed AN, AlHarbi NG. The association of metformin use with vitamin B12 deficiency and peripheral neuropathy in Saudi individuals with type 2 diabetes mellitus. PLoS One. 2018;13:e0204420.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 18]  [Cited by in RCA: 46]  [Article Influence: 6.6]  [Reference Citation Analysis (0)]
35.  Moore E, Mander A, Watters D. More adverse neurological consequences of metformin-induced vitamin B12 deficiency. South Med J. 2011;104:541-2; author reply 542.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in RCA: 1]  [Reference Citation Analysis (0)]
36.  Karedath J, Batool S, Arshad A, Khalique S, Raja S, Lal B, Anirudh Chunchu V, Hirani S. The Impact of Vitamin B12 Supplementation on Clinical Outcomes in Patients With Diabetic Neuropathy: A Meta-Analysis of Randomized Controlled Trials. Cureus. 2022;14:e31783.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in RCA: 1]  [Reference Citation Analysis (0)]
37.  Didangelos T, Karlafti E, Kotzakioulafi E, Giannoulaki P, Kontoninas Z, Kontana A, Evripidou P, Savopoulos C, Birkenfeld AL, Kantartzis K. Efficacy and Safety of the Combination of Palmitoylethanolamide, Superoxide Dismutase, Alpha Lipoic Acid, Vitamins B12, B1, B6, E, Mg, Zn and Nicotinamide for 6 Months in People with Diabetic Neuropathy. Nutrients. 2024;16:3045.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in RCA: 2]  [Reference Citation Analysis (0)]
38.  Sands T, Jawed A, Stevenson E, Smith M, Jawaid I; Guideline Committee. Vitamin B(12) deficiency: NICE guideline summary. BMJ. 2024;385:q1019.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 3]  [Cited by in RCA: 6]  [Article Influence: 6.0]  [Reference Citation Analysis (0)]
39.  American Diabetes Association Professional Practice Committee. Introduction and Methodology: Standards of Care in Diabetes-2025. Diabetes Care. 2025;48:S1-S5.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 7]  [Cited by in RCA: 7]  [Article Influence: 7.0]  [Reference Citation Analysis (0)]
40.  Handelsman Y, Bloomgarden ZT, Grunberger G, Umpierrez G, Zimmerman RS, Bailey TS, Blonde L, Bray GA, Cohen AJ, Dagogo-Jack S, Davidson JA, Einhorn D, Ganda OP, Garber AJ, Garvey WT, Henry RR, Hirsch IB, Horton ES, Hurley DL, Jellinger PS, Jovanovič L, Lebovitz HE, LeRoith D, Levy P, McGill JB, Mechanick JI, Mestman JH, Moghissi ES, Orzeck EA, Pessah-Pollack R, Rosenblit PD, Vinik AI, Wyne K, Zangeneh F. American association of clinical endocrinologists and American college of endocrinology - clinical practice guidelines for developing a diabetes mellitus comprehensive care plan - 2015. Endocr Pract. 2015;21 Suppl 1:1-87.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 123]  [Cited by in RCA: 277]  [Article Influence: 27.7]  [Reference Citation Analysis (0)]
41.  Tesfaye S, Kempler P. Conventional management and current guidelines for painful diabetic neuropathy. Diabetes Res Clin Pract. 2023;206 Suppl 1:110765.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in RCA: 2]  [Reference Citation Analysis (0)]
42.  Diabetes Canada Clinical Practice Guidelines Expert Committee; Bril V, Breiner A, Perkins BA, Zochodne D. Neuropathy. Can J Diabetes. 2018;42 Suppl 1:S217-S221.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 17]  [Cited by in RCA: 29]  [Article Influence: 4.1]  [Reference Citation Analysis (0)]
43.  Atmaca A, Ketenci A, Sahin I, Sengun IS, Oner RI, Erdem Tilki H, Adas M, Soyleli H, Demir T. Expert opinion on screening, diagnosis and management of diabetic peripheral neuropathy: a multidisciplinary approach. Front Endocrinol (Lausanne). 2024;15:1380929.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 2]  [Cited by in RCA: 10]  [Article Influence: 10.0]  [Reference Citation Analysis (0)]
44.  Didangelos T, Karlafti E, Kotzakioulafi E, Margariti E, Giannoulaki P, Batanis G, Tesfaye S, Kantartzis K. Vitamin B12 Supplementation in Diabetic Neuropathy: A 1-Year, Randomized, Double-Blind, Placebo-Controlled Trial. Nutrients. 2021;13:395.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Full Text (PDF)]  [Cited by in Crossref: 44]  [Cited by in RCA: 64]  [Article Influence: 16.0]  [Reference Citation Analysis (0)]
45.  Vidal-Alaball J, Butler CC, Cannings-John R, Goringe A, Hood K, McCaddon A, McDowell I, Papaioannou A. Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 2005;CD004655.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 61]  [Cited by in RCA: 89]  [Article Influence: 4.5]  [Reference Citation Analysis (0)]
46.  Devalia V, Hamilton MS, Molloy AM; British Committee for Standards in Haematology. Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol. 2014;166:496-513.  [RCA]  [PubMed]  [DOI]  [Full Text]  [Cited by in Crossref: 205]  [Cited by in RCA: 277]  [Article Influence: 25.2]  [Reference Citation Analysis (0)]