Background: Cardiovascular disease (CVD) is a leading cause of mortality in individuals with type 1 diabetes mellitus (T1DM), with conventional risk factors not fully explaining the heightened risk. Study Design: We conducted a cross-sectional study comparing serum protein levels in 134 participants with T1DM from the Coronary Artery Calcification in Type 1 Diabetes study to 47 healthy controls, followed by a case-cohort study among 181 T1DM participants, including 47 with CVD events. Linear regression analyzed serum protein levels by diabetes status, while Cox proportional hazards regression assessed associations with incident CVD. Findings: Among 28 serum proteins tested, five showed significant differences between T1DM patients and controls, with alpha-2-macroglobulin (A2M) being notably 1.6-fold higher in T1DM. Adjusted models revealed that A2M (mean hazard ratio [HR] 2.5, 95% confidence interval [CI] 1.5-4.1) and apolipoprotein C3 (APOC3, mean HR 1.7, 95% CI 1.1-2.7) were independently associated with incident CVD in T1DM. Conclusions: Elevated serum levels of A2M in T1DM suggest its potential role as a marker—and possibly a mediator—of CVD risk in these patients, alongside APOC3. These findings underscore the significance of specific serum proteins in understanding and managing CVD risk in T1DM.

The targeted PRM MS data of peptides from each serum protein were analyzed using Skyline (version 23.1.0.380), an open-source program. Before digestion, we added an equal amount of 15N-labeled APOA1 to each serum sample as an internal standard. The peak areas of all the transitions of a peptide detected by PRM were summed to get the total peak area for the peptide. Transitions with interferences were omitted from the analyses. To normalize the peak area of a peptide, the total peak area of all selected transitions of the peptide was divided by the peak area of each of the three 15N-labeled peptides from 15N-APOA1, and the ratios were used for quantification. To calculate relative levels of a peptide, we first calculated the average ratio of that peptide to one of the three 15N-APOA1 peptides in T1DM subjects without CVD, then the level of each subject was divided by that average ratio, with the new ratio representing the relative level of each subject. Next, the three new ratios (from comparison to three 15N-labeled peptides from 15N-APOA1) were averaged. If two or more peptides were quantified for a protein, their relative levels were averaged to obtain the relative level of that protein in serum.

The current study contains two study designs. The first study, which investigated the association between serum proteins and T1DM status, was a cross-sectional study with 134 randomly selected participants with T1DM (without incident CVD) and 47 healthy controls without diabetes or incident CVD from the CACTI study. The second study, which investigated the associations between serum proteins and incident CVD in T1DM participants, was a case-cohort study, in which 145 participants in a randomly selected sub-cohort of T1DM participants (including 11 with incident CVD) and all 47 T1DM subjects with incident CVD events. Serum samples from the 181 participants with T1DM in the second study were used in previous studies. The cases were 47 CACTI subjects with incident CVD, and the cohort was 145 randomly selected subjects (including 134 T1DM subjects without incident CVD and 11 incident CVD case subjects). Samples from all 238 subjects were de-identified, and MS/MS analyses were blinded.