Table 2.

Optimizing measurements of proportional elevations of prohormones in type 1 diabetes

Factor	Important considerations
Timing of sampling	• Samples from all participants should be optimally obtained at consistent timing relative to fasting vs. glucose or mixed-meal stimulation.
	• For normalization, prohormones and mature hormones should be measured in samples obtained from the same time point.
	• Fasting PI/C ratios reportedly elevated in individuals at risk for or diagnosed with type 1 diabetes.
	• Stimulated PI/C ratios may be less impacted relative to fasting values in longer-duration type 1 diabetes, but definitive understanding of timing of peak PI/C ratios in these populations requires more robust study that includes multiple stimulated time points.
Prohormone measurements
• Proinsulin assay	• Commercially available assays include radioimmunoassays and ELISA.
	• Does assay of interest test intact (unprocessed) proinsulin or total proinsulin (unprocessed + different combinations of partially processed split products depending on assay)? Cross-reactivity with partially processed proinsulin species is common in most commercial proinsulin and insulin assays. No commercial assays are currently available that quantify des-64,65 proinsulin or des-31,32 proinsulin individually.
	• Antibody-based assays are well established but often not well characterized. Because standards for partially processed forms of proinsulin are not widely available for external validation, there is uncertainty regarding which proinsulin products commercially available antibodies recognize.
	• Has assay of interest been externally validated to show reproducible results?
	• Is assay of interest calibrated based on 84/611 (older) or 09/296 (newer) intact proinsulin standard (60)? Due to diminishing supply, in 2014 the original World Health Organization intact proinsulin standard was switched to the 09/296 standard (60). Because of this, although newer intact and total proinsulin assays are calibrated based on the 09/296 standard, several older assays are calibrated against the prior 84/611 standard, thereby limiting direct comparison of results, given that values obtained with assays based on different standards are not quantitatively equivalent.
	• While partially processed split products do not add discriminatory capability above that of intact proinsulin for samples from control subjects and patients with type 2 diabetes, this has not been tested in type 1 diabetes (47).
	• Targeted MS-based techniques may allow for antibody-independent measurements and validation of antibody-based assays. But such assays are still in the developmental stage at research laboratories.
• ProIAPP assay	• Externally validated proIAPP1–48 ELISA is available through research laboratories only (6).
	• Assays for intact proIAPP1–67 and other intermediate forms are currently under development.
	• Targeted MS-based techniques are currently under development.
Mature hormone measurements	• Understanding of proportional elevations in prohormones requires careful quantification of levels relative to mature hormones. Thus, optimizing mature hormone measurement is also a key goal.
• C-peptide or insulin assay	• Elevations in both PI/C and proinsulin-to-insulin ratios have been described in circulation for certain populations relevant to type 1 diabetes (7,28,29,61).
	• PI/C ratio outperforms proinsulin-to-insulin ratio to predict incident diabetes in insulin-resistant populations, as circulating insulin values can reflect altered hepatic insulin clearance (26).
	• Measurement of endogenous insulin levels may be confounded by use of insulin analogs.
	• International standards are available for both C-peptide and insulin.
	• Certain immunoassays for C-peptide and insulin may cross-react with proinsulin species.
	• MS-based based assays for C-peptide and intact insulin are available through commercial laboratories (e.g., Quest Diagnostics).
• IAPP assay	• Commercially available assays include radioimmunoassays and ELISA.
	• Commercial assays have not been externally validated.
	• Externally validated immunoassay for mature, C-terminally amidated IAPP is available through research laboratories (5).
	• No international standard exists for cross calibration of assays.
	• MS-based techniques are currently under development.