Early detection of metabolic dysregulation using water T₂ analysis of biobanked samples

The ability to use frozen biobanked samples from cohort studies and clinical trials is critically important for biomarker discovery and validation. Here we investigated whether plasma and serum water transverse relaxation times (T₂) from frozen biobanked samples could be used as biomarkers for metabolic syndrome (MetS) and its underlying conditions, specifically insulin resistance, dyslipidemia, and subclinical inflammation.

Plasma and serum aliquots from 44 asymptomatic, non-diabetic human subjects were biobanked at -80°C for 7-9 months. Water T₂ measurements were recorded at 37°C on 50 µL of unmodified plasma or serum using benchtop nuclear magnetic resonance relaxometry. The T₂ values for freshly drawn and once-frozen-thawed ("frozen") samples were compared using Huber M-values (M), Lin concordance correlation coefficients (ρ_c), and Bland-Altman plots. Water T₂ values from frozen plasma and serum samples were compared with >130 metabolic biomarkers and analyzed using multi-variable linear/logistic regression and ROC curves.

Frozen plasma water T₂ values were highly correlated with fresh (M=0.94, 95% CI 0.89, 0.97) but showed a lower level of agreement (ρ_c=0.74, 95% CI 0.62, 0.82) because of an average offset of -5.6% (-7.1% for serum). Despite the offset, frozen plasma water T₂ was strongly correlated with markers of hyperinsulinemia, dyslipidemia, and inflammation and detected these conditions with 89% sensitivity and 91% specificity (100%/63% for serum). Using optimized cut points, frozen plasma and serum water T₂ detected hyperinsulinemia, dyslipidemia, and inflammation in 23 of 44 subjects, including nine with an early stage of metabolic dysregulation that did not meet the clinical thresholds for prediabetes or MetS.

Plasma and serum water T₂ values from once-frozen-thawed biobanked samples detect metabolic dysregulation with high sensitivity and specificity. However, the cut points for frozen biobanked samples must be calibrated independent of those for freshly drawn plasma and serum.