Ethical approval for this study was received from the University of Calgary Centre for Advancement of Health. From December 2002 until May 2007, patients with preexisting type 2 diabetes and a primary diagnosis of peripheral neuropathy were assessed within the Neuromuscular Clinic at the University of Calgary. These patients then underwent further clinical, laboratory, and electrophysiological evaluation of their neuropathy. The presence of diabetes was verified by two separate positive results: two prior fasting glucose results of ≥7.1 mmol/l (126 mg/dl) or two oral glucose tolerance tests leading to a 2-h serum glucose of ≥11.1 mmol/l (200 mg/dl) (based on Canadian Diabetes Association guidelines). The age of diagnosis of diabetes and the duration of symptoms of DPN were recorded. History of other systemic illnesses, alcohol use, toxin and medication exposures, and family history of neuropathy was documented to assess for other potential causes of peripheral neuropathy. The duration of metformin therapy and dosage history were determined for each patient by review of their medical record, and these dosages were confirmed verbally by the patients; these data were used to calculate a cumulative lifetime dose of metformin for each patient. Use of other antidiabetic agents was recorded.
All patients underwent laboratory testing including a complete blood count, electrolytes, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltranspeptidase, alkaline phosphatase, albumin, total bilirubin, international normalized ratio, thyroid-stimulating hormone, erythrocyte sedimentation rate, antinuclear antibody, extracted nuclear antibody, serum protein electrophoresis, rheumatoid factor, lactate, and serum folate. A1C was measured in all patients. The sensitivity for detection of gammopathy in our center is 2 g/l by serum protein electrophoresis, with immunofixation performed when peaks are found in the range of 2–4 g/l. Serum Cbl levels were measured by cobas e immunoassay analysis (Roche Diagnostics), Hcy levels were measured using high-performance liquid chromatography, and MMA levels were measured using mass spectrometry for all patients. The lower limit of normal for Cbl in our center is 210 pmol/l (285 pg/ml). The upper limit for Hcy is 13.7 μmol/l (1.85 mg/l) in adult men, 9.9 μmol/l (1.34 mg/l) in adult women aged ≤49 years and 12.8 μmol/l (1.73 mg/l) in adult women aged >49 years; here we adopt a conservative upper limit of 13.7 μmol/l (1.85 mg/l) for all participants. The upper limit of normal for MMA in our center is 0.15 μmol/l. All blood testing was performed by Calgary Laboratory Services.
Patients were excluded if potential causes for peripheral neuropathy other than diabetes and Cbl deficiency were identified, if they had previously been treated with metformin and had discontinued therapy, if they had received <6 months of metformin treatment at the time of assessment, if they had impaired glucose tolerance only, or if they had juvenile onset of diabetes or a frank requirement for insulin at diagnosis (i.e., possible type 1 diabetes). Last, patients were excluded if they refused concurrent electrophysiological or laboratory testing. We did not exclude patients with renal failure concurrently using metformin, although renal failure is often considered a contraindication to metformin use because of the potential for lactic acidosis; however, many patients do not discontinue metformin use after diagnosis of renal impairment (19
), and lactate levels were normal in all patients.
Complete standardized neurological examinations were performed in all patients with DPN, including tone, power, deep tendon reflexes, sensory function, Romberg testing, gait, and tandem gait. Tandem gait was recorded as the number of heel to toe steps along a straight line the patient could perform to a normal threshold value of 6. Each patient was also scored using the Toronto Clinical Scoring System (TCSS) (20
) by an unblinded investigator before knowledge of laboratory results. The TCSS was developed as a clinical screening tool for the presence and severity of DPN that emphasizes sensory deficits. Although it introduces some subjectivity in scoring, it has been validated by sural nerve fiber density. We also determined the Neuropathy Impairment Score (NIS), a scale scoring weakness of groups of muscles of the head and neck, upper limbs, and lower limbs; tactile, vibratory, and joint position sensation; pinprick sensation of index fingers and great toes; and reflexes, for each patient (21
Electrophysiological assessment was performed after clinical assessment and before knowledge of the laboratory results using a Dantec Datapoint (Dantec Dynamics, Bristol, U.K.). Sensory and motor nerves of the nondominant upper and lower extremities were tested within 3 months of clinical assessment. Motor nerve conduction studies (NCSs) were performed using stimulation of the median nerve (wrist and elbow), ulnar nerve (wrist, below elbow, and above elbow), peroneal nerve (ankle and below fibular head and above fibular head locations), and tibial nerve (ankle and popliteal fossa locations). For each motor nerve, distal motor latencies, compound motor action potentials, and conduction velocities were obtained or calculated. F wave latencies were obtained from median, ulnar, peroneal, and tibial nerves. Sensory NCSs were performed using the median (digits 2 and 4), ulnar (digits 4 and 5), superficial radial, superficial peroneal, and sural nerves with sensory nerve action potentials (SNAPs), onset latency, and conduction velocity obtained or calculated. Temperatures were maintained at ≥32°C for the upper extremities and ≥30°C for the lower extremities during NCS testing. Although all participants completed electrophysiological testing, some participants do not have complete data for all individual nerves.
After all clinical, electrophysiological, and laboratory testing, monthly intramuscular Cbl was prescribed for those patients with abnormal Cbl, MMA, or Hcy levels. Sural nerve biopsies were done only in clinical situations when vasculitis or another serious cause of peripheral neuropathy was suspected.
Group equivalence for patient age, duration of type 2 diabetes, duration of peripheral neuropathy symptoms, A1C, and alcohol exposure were compared by independent-samples t
tests; sex and proportion using other antidiabetic agents were compared by χ2
test. Elements of the past medical history (e.g., iron deficiency anemia and hereditary spherocytosis) were broadly classified (e.g., hematological disease) and are summarized in ; these were not compared statistically because of their heterogeneity. These elements of the history are detailed in the appendix (available online at http://care.diabetesjournals.org/cgi/content/full/dc09-0606/DC1
). The primary outcome measures were Hcy, MMA, and Cbl blood levels, clinical neuropathy severity (TCSS and NIS), and electrophysiological markers of neuropathy; of the latter, we chose to test sensory NCS in the lower extremity (conduction velocity and SNAP for superficial peroneal and sural nerves), as we felt these would be most in keeping with exacerbation due to Cbl deficiency. These data did not follow a normal distribution (by Shapiro-Wilk test), and comparisons were made using a Mann-Whitney U
test. Proportions of patients with deficiency of Cbl and upregulation of Hcy and MMA were compared using χ2
tests. Bivariate correlations of clinical and laboratory variables with cumulative metformin dose were calculated using a Spearman ρ test. Last, a linear regression analysis was performed using the NIS total score as the dependent variable and age, duration of diabetes, A1C, and presence of metformin exposure as explanatory variables.