DOI: 10.5061/dryad.rxwdbrv7w
title: Lens fluorescence and skin fluorescence in the Copenhagen Twin Cohort Eye Study: Covariates and heritability

description: Lens and skin fluorescence are related to the systemic accumulation of
 advanced glycation end products, which is accelerated in diabetes. We have
 examined lens fluorescence and skin fluorescence in healthy adult twins.
 The study enrolled twins aged median 59 years from a national
 population-based registry. Diabetic individuals were excluded from
 analysis. The interrelatedness between fluorescence parameters and
 relations between fluorescence and age, current HbA1c and smoking pack
 years were examined using correlation tests and mixed model linear
 regression analyses. Broad-sense heritability was analyzed and compared
 for lens fluorescence, skin fluorescence and HbA1c. Lens fluorescence and
 skin fluorescence were crudely interrelated (R = 0.38). In linear
 regression analyses, age explained a larger fraction of the variance in
 lens fluorescence (R2 = 32 %) than in skin fluorescence (R2 = 20 %),
 whereas HbA1c explained smaller variance fractions (R2 = 3 % and 8 %,
 respectively) followed by smoking pack years (4 % and 3 %, respectively).
 In multivariate analyses, age, HbA1c and smoking pack years combined
 explained more of the variance in lens fluorescence (R2 = 35 %) than in
 skin fluorescence (R2 = 21 %), but the influence of HbA1c on lens
 fluorescence was not statistically significant (p = .2). Age-adjusted
 broad-sense heritability was 85 % for lens fluorescence, 53 % for skin
 fluorescence and 71 % for HbA1c in best fitting heritability models. Both
 fluorescence parameters increased with age, current glycemia and
 cumulative smoking. Lens fluorescence was found to be a predominantly
 heritable trait, whereas skin fluorescence was more influenced by
 environmental factors and closer related to current glycemia. The results
 suggest that skin fluorophores have a faster turn-over than lens
 fluorophores.
description: Data sources: Lens fluorescence was measured
 in the right eye of phakic subjects using a commercial ocular fluorometer
 (Fluortron Master TM-2 with Windows software, revision B.17, OcuMetrics,
 Mountain View, California, USA), approximately 1 hour after dilation with
 tropicamide 1% eye drops. The device measures blue-green fluorescence at
 incremental steps of 0.125 mm along the optical axis of the eye using
 excitation light at 430-490 nm and detection at 530-630 nm with results
 reported in units of equivalent fluorescein concentration in water.
 Measurements were performed under scotopic lighting conditions.
 Absorption-corrected anterior lens peak fluorescence was calculated using
 the manufacturer’s software. Lens fluorescence peak values were corrected
 for ambient light in the examination room by subtraction of the lowest
 fluorescence readings in each scan (averaged from the fluorescence
 intensities of the 15/148 steps with the lowest intensities). Three
 successful scans were attempted for each subject. Unsuccessful scans
 counted scans with ambient background light values above 30% of the
 posterior absorption-corrected lens peak fluorescence, as recommended by
 the manufacturer, and if blinking had occurred at critical points during
 the scan. Lens fluorescence values used for data analysis were based on
 the average absorption-corrected anterior peak value of three successful
 scans. Study subjects in whom three successful scans could not be obtained
 were omitted from analysis. Skin fluorescence was measured on the anterior
 forearm with a designated commercial device (Diagnoptics AGE Reader,
 Diagnoptics Technologies B.V., Groningen, Netherlands). The instrument
 emits light at 300-420 nm, with peak intensity at 370 nm, on a 4
 cm<sup>2</sup> skin area and measures emission at 300-600 nm.
 Data output is a double-digit arbitrary unit (AU) index of 420-600 nm
 fluorescence relative to reflected 300-420 nm emission multiplied by 100.
 The average of three readings per subject was used for analyses. According
 to the manufacturer’s guidelines, subjects with excessive sweating,
 tattoos, recently applied skin cream or recent intensive sunbathing
 affecting the skin region of interest were excluded from analysis. The
 first measurement after device start-up was routinely discarded, as
 recommended by the manufacturer. We choose to include
 three lens fluorescence and three skin fluorescence readings per subject
 since three are enough to evaluate reproducibility of measurements and the
 maximum to which one can reasonably expose a study participant in a study
 that also includes other procedures. Also, unilateral measurements only do
 not require statistical adjustments due to paired organ data
 dependencies. Blood samples obtained during the
 examinations were analyzed for HbA<sub>1c</sub> . Data on
 accumulated smoking pack years were obtained by interview. Participants
 who reported &lt;1 pack year were categorized as
 non-smokers. Statistical analyses:
 Microsoft Excel 360 for Windows 10 was used for demographic
 statistics, GraphPad Prism v9.0.0.121 for reproducibility of measurements
 analyses and R-Studio v1.2.5001 for Windows 10 was used for all other
 statistical analyses. Normality was tested by Shapiro-Wilk normality
 tests. Fluorescence parameters were transformed by log10 to obtain normal
 distributions. All fluorescence values reported have been back-transformed
 to geometric mean values with 95% confidence interval. Non-parametric
 parameters were reported in medians and inter-quantile ranges.
 Reproducibility of fluorescence measurements were assessed by three-group
 one-way ANOVA analyses of log10-transformed data. Pearson’s correlation
 tests were used in case of normal distributions and Spearman’s rank
 correlation tests were used for non-normally distributed parameters.
 Univariate and multivariate log-level linear mixed model regression
 analyses adjusted for twin-pair dependencies were performed with the R
 functions ‘lmer()’ (lme4 v.1.1.26 package) and ‘modelTest ()’ (JWileymisc
 v. 1.2.0 package). Reported coefficient estimates from regression analyses
 were transformed by antilog to designate percentage change in fluorescence
 per unit change in either age (years), smoking (pack years) or
 HbA<sub>1c</sub> (mmol/mol),. Broad-sense heritability was
 calculated for lens fluorescence, skin fluorescence and
 HbA<sub>1c</sub> using a linear regression model of each
 parameter as a function of age with the R function ‘twinlm()’ (mets v.
 1.2.8.1 package). Best-fit models were found by Akaike’s information
 criterion (AIC). The lowest AIC-value defined the best fitting model for
 lens fluorescence, skin fluorescence and HbA<sub>1c</sub>, but
 models with AIC-values between the value of the best fitting model and the
 value of the best model plus two units were considered non-inferior to
 true best fitting models.
description: Please review the spreadsheet tab "ReadMe_tab" for
 usage notes.