Skin collagen fluorophore LW-1 versus skin fluorescence as markers for the long-term progression of subclinical macrovascular disease in type 1 diabetes

Abstract

Background: Skin collagen Long Wavelength Fluorescence (LWF) is widely used as a surrogate marker for accumulation of advanced glycation end-products. Here we determined the relationship of LWF with glycemia, skin fluorescence, and the progression of complications during EDIC in 216 participants from the DCCT.

Methods: LW-1 and collagen-linked fluorescence (CLF) were measured by either High Performance Liquid Chromatography (HPLC) with fluorescence detection (LW-1) or total fluorescence of collagenase digests (CLF) in insoluble skin collagen extracted from skin biopsies obtained at the end of the DCCT (1993). Skin intrinsic fluorescence (SIF) was noninvasively measured on volar forearm skin at EDIC year 16 by the SCOUT DS instrument.

Results: LW-1 levels significantly increased with age and diabetes duration (P < 0.0001) and significantly decreased by intensive vs. conventional glycemic therapy in both the primary (P < 0.0001) and secondary (P < 0.037) DCCT cohorts. Levels were associated with 13-16 year progression risk of retinopathy (>3 sustained microaneurysms, P = 0.0004) and albumin excretion rate (P = 0.0038), the latter despite adjustment for HbA1c. Comparative analysis for all three fluorescent measures for future risk of subclinical macrovascular disease revealed the following significant (P < 0.05) associations after adjusting for age, diabetes duration and HbA1c: coronary artery calcium with SIF and CLF; intima-media thickness with SIF and LW-1; and left ventricular mass with LW-1 and CLF.

Conclusions: LW-1 is a novel risk marker that is robustly and independently associated with the future progression of microvascular disease, intima-media thickness and left ventricular mass in type 1 diabetes. Trial registration NCT00360815 and NCT00360893 at clinicaltrials.gov.

Figures

Fig. 1

Unadjusted LW-1 vs. age in skin collagen digests of DCCT patients. For all graphs, regression lines and 95 % CI prediction for nondiabetic controls are shown. a Unadjusted LW-1 expressed as a percentage of total fluorescence at ex/em 348/463 nm in skin collagen digests vs. age. Percent LW-1 was determined by manually collecting the LW-1 peak by HPLC and comparing its fluorescence yield with that of the digest itself at wavelengths same as described in the “Methods” section. b and c Unadjusted LW-1 levels vs. age at DCCT closeout in the primary and secondary cohorts. Regression lines: a y = 3.4 + 0.172x, r = 0.48, P = 0.002, n = 39. b and c y = 1.8 + 6.6x, r = 0.64, P < 0.0001, n = 42. Treatment (cohort) circle, conventional (primary); triangle, conventional (secondary); filled circle, intensive (primary); filled triangle, intensive (secondary)

Fig. 2

The effect of intensive vs. conventional glycemic therapy on LW-1 levels after adjustment for age and diabetes duration in the primary and secondary DCCT cohorts at DCCT closeout. LW-1 responds significantly to lowering of glycemia after adjusting for age and duration of diabetes (nondiabetic controls: age-adjusted). Each bar represents the mean ± SD. Means with a different letter superscript are significantly different (P ≤ 0.0001)

Fig. 3

Plots of unadjusted specific intrinsic fluorescence (SIF by SCOUT DS) vs. unadjusted LW-1 (left) and unadjusted collagen-linked fluorescence/CLF (right) in DCCT/EDIC participants (see “Methods”). LW-1 and CLF were assayed in enzymatic digests of insoluble collagen prepared from skin biopsies obtained from the buttock region of participants at DCCT closeout (1993). SIF was obtained by noninvasive measurements of autofluorescence made by the SCOUT DS on the underside of the left forearm skin near the elbow for most DCCT participants at EDIC years 16–17 (2009–2010). Regression line and 95 % CI prediction: CLF y = 141 + 2.1x, r = 0.209, P = 0.004, n = 185; LW-1: y = 195 + 8.9x, r = 0.25, P = 0.001, n = 185. Symbols used: see Fig. 1

Fig. 4

Univariate regression models of complications vs. age- and duration-adjusted LW-1 levels (mean ± SD) across treatment groups at DCCT closeout. MA microaneurysms, AER albumin excretion rates, NCV/ANS nerve conduction velocity/autonomic nervous system, NS nonsignificant (P > 0.05). Analysis was limited to those with no respective complication at DCCT baseline (excluding 10 subjects with AER > 40, 19 with confirmed clinical neuropathy, and the secondary cohort for sustained ≥3 MA). Indices of retinopathy, microaneurysms, nephropathy and neuropathy were quantified as previously described [19]. ‡ P value is from a Likelihood Ratio test (LRT) between the respective reduced model and the full model

Fig. 5

Spearman correlations for markers of skin fluorescence vs. coronary artery calcification (CAC). Markers were adjusted for age and diabetes duration. CAC was determined at EDIC years 7–9 by computed tomography as scores in Agatson units

Fig. 6

Linear plots of intima-media thickness (IMT) as well as the change in IMT between EDIC years 1–6 vs. markers for skin fluorescence (LW-1, SIF), as indicated. Preliminary Spearman correlation analysis showed that LW-1 and SIF (but not CLF) significantly (P 1c (P = 0.013) and borderline nonsignificantly with diabetes duration (P = 0.068). Thus, all variables in these graphs have been adjusted for age, diabetes duration and HbA1c (both DCCT and EDIC). For all graphs, the regression line and 95 % CI prediction of data points are shown: (left) IMT = 5.64 + 0.001 (LW-1), n = 127; (middle) IMT = 5.68 + 0.028 (SIF), n = 121; (right) change in IMT between EDIC year 1 and 6, ∆IMT = 0.001(LW-1)–0.339, n = 121. All values for IMT and ∆IMT have been multiplied by 10. For each graph, the correlation (r) and significance (P) are inserted. Symbols used: see Fig. 1

Fig. 7

Linear plots of left ventricular mass (LVM) at EDIC years 14–16 vs. LW-1 and CLF, as indicated. Preliminary Spearman correlation analysis showed that both LW-1 and CLF were significantly (P  0.05). LW-1 and CLF were adjusted for age, diabetes duration and HbA1c (both DCCT and EDIC). However, LVM did not significantly correlate with any of these latter variables, thus no adjustments were made (P = 0.27, P = 0.98, P > 0.25, respectively). For both graphs (n = 142), the regression line and 95 % CI prediction are shown: (left) LVM = 59 + 0.021 (LW-1); (right) LVM = 59 + 0.081 (CLF). For each graph, the correlation (r) and significance (P) are inserted. Symbols used: see Fig. 1