Study design

The DPP and DPPOS design, eligibility, and baseline characteristics have been reported elsewhere (26, 27). The DPP enrolled 3234 participants with impaired glucose tolerance and fasting blood glucose of 95 to 125 mg/dL (≤125 mg/dL in American Indians) who were at least 25 years of age and had body mass index (BMI) of 24 kg/m² or higher (≥22 kg/m² in Asian Americans). Enrollment began in July 1996 and ended in May 1999. Participants were randomly assigned to one of three treatments: placebo, an intensive lifestyle program, or metformin 850 mg twice daily. All of the 27 DPP clinical study centers as well as the DPP Coordinating Center had institutional review board approval. All participants gave written informed consent.

Mean follow-up at the end of the DPP was 3.2 years. At the end of the DPP, all participants were offered a group-implemented lifestyle intervention and invited to enroll in the follow-up study, DPPOS. During the DPPOS, those originally assigned to metformin received open-label metformin at the same prior dose of 850 mg twice daily, which was continued until such time as diabetes developed and HbA1c value reached ≥ 7%. At this point, study metformin was discontinued, and the participant referred to his or her own physician for treatment of diabetes, which may have included metformin. Samples used for measurement of vitamin B12 and homocysteine, as a corroborating indicator of vitamin B12 deficiency, were collected an average of 5 years (DPPOS year 1) and 13 years (DPPOS year 9) after initial randomization and analyzed in 2011 (Supplemental Data). Stored serum samples from other time points, including the DPP baseline, were not available. DPPOS year 9 was chosen to coincide with planned neuropathy outcome measures.

Vitamin B12 levels were measured using Tosoh reagent on a Tosoh 1800 analyzer (TOSOH Bioscience, Inc). The assay sensitivity is 50 pg/mL, and the results are traceable to the World Health Organization IS 03/178 reference material. The interassay coefficients of variation obtained on quality control samples with high-, medium- and low levels ranged from 7.2% to 8.8%. Homocysteine levels were measured by a homogeneous enzymatic assay on a Roche Modular P analyzer (Roche Diagnostics, Inc., Indianapolis, IN). The assay sensitivity is 1 μmol/L and the results are traceable to the National Institute of Standards and Technology reference preparation homocysteine. The interassay coefficients of variation obtained on quality control samples with high-, medium-, and low levels ranged from 3.9 to 8.9%.

Hemoglobin and hematocrit were obtained as a safety measure to detect B12 deficiency anemia in placebo and metformin-assigned participants annually in DPP and in DPPOS year 1, but only in participants actively taking study-provided metformin thereafter. Participants identified with anemia were referred to their own physicians for evaluation and treatment.

Low vitamin B12 was defined as ≤ 203 pg/mL, and borderline-low levels were defined as between 204 and 298 pg/mL, inclusive (28, 29). Anemia was defined as hemoglobin < 12 g/dL or hematocrit < 36% (females) and hemoglobin < 13 g/dL or hematocrit < 40% (males). Elevated homocysteine was defined as > 13.7 μmol/L (13). Metformin exposure, documented by counting returned tablets at each study visit, was assessed semiannually. Study-provided metformin usage was calculated, as well as total metformin years of exposure, which includes both protocol-based and nonprotocol metformin use (ie, prescribed outside of the study for diabetes treatment). Clinical neuropathy was defined as a reduction or absence of light touch sensation to monofilament (Semmes-Weinstein, 10 g) in either foot (< eight of 10 applications detected). The Michigan Neuropathy Screening Instrument (MNSI) was conducted annually and was analyzed for DPPOS years 1 and 9, concurrent with B12 measurements (30).