Optical Signatures of Diabetes Mellitus in Skin
1
Ediger , Edward
1
Hull ,
2
Baynes ,
Marwood N.
L.
John W.
1
1
Regina Trujillo , and Amelia H.T. Unione
1InLight Solutions, 800 Bradbury SE,
2 Dept
of Chemistry and Biochemistry
University of South Carolina, Columbia, SC 29208
Albuquerque NM, 87106
Introduction
Principal Components Analysis
Spectroscopic Results
Variance explained = 98.9%
Spectroscopic & chemical basis for optical
diabetes screening not fully established
Control
Hyperglycemic
Anaerobic
Control
Hyperglycemic
Anaerobic
2
PCA Scores - Factor 1
PCA Factor 1
NIR reflectance mode - absorption spectra
0.25
0.2
0.15
Water bands
0.1
Collagen
features
0.05
320
340
360
4500
5000
5500
6000
6500
7000
4500
5000
Wavenumbers (cm -1)
5500
6000
6500
7000
Wavenumbers (cm -1)
NIR spectra were acquired in transmission (left) and reflectance (right) modes. Each trace (# of traces = 120) depicts the average NIR absorption spectrum
for a particular specimen (# of specimens = 12) for a given spectroscopy session (# of data acquisition sessions = 10) covering the 5 week study. In each
session, multiple 60-second acquisitions (transmission = 8 & reflectance = 6) were made for each specimen. For each 60 second acquisition, the FTIR
instrument collects ~170 individual spectra. Hence, each trace represents the average of numerous spectra (transmission = 1360; reflectance = 1020)
collected from a particular collagen specimen during a single data session. Spectra are color coded to denote in which incubation media they resided.
1
2
3
4
5
6
7
6
pcritical
Specimens subdivided and placed into 1 of 3
incubation media:
2
de 500
ch xtro mM
el se
at
or +
s
Hyperglycemic, anaerobic
*
*
*
*
4
2
 = 315-385nm
x
m = 400nm
Transmission
 = 325-445nm
x
m = 460nm
 = 325nm
x
m = 340-500nm
Fluorescence
The upper figures illustrate representative results from the PCA decomposition of a spectral data set. The 1st PCA factor for the excitation spectra (x = 315 –
385nm; m = 400nm) is shown in the upper left panel. This spectral shape accounts for 98.9% of the variance in that data set. In the upper right panel, the
PCA scores for the 1st factor are plotted vs the respective pentosidine concentrations. The specimens are color-coded by incubation medium and the
symbols denote the specimens (circles = 1, x = 2, squares = 3 & triangles = 4). PCA decomposition was performed on all data sets. An Ordinary Least
Squares (OLS) fit was performed by regressing the PCA scores on the AGE concentrations (pentosidine) after removing global time trends. The significance
of the score/AGE relationship for individual PCA factors (-log(p)) are plotted in the lower panel. The number of factors presented for each spectral set
accounts for 99.95% of the spectroscopic variance in that set. The critical value (p = 0.01) is indicated by the dotted line. The bars noted with * represent pvalues that were less than the sensitivity of the calculation (p=0.001) and were thus set to 0.0005.
Partial Least Squares
Standard Error of Prediction
0.35
SEP (pmol/mg protein)
Hyperglycemic
Intensity (arb units)
Normoglycemic
* * *
Fluorescence
x = 325 – 445 nm; m = 460nm
0.3
0.25
0.2
0.15
AGE assay method*
0
2
4
6
8
10
Pentosidine Prediction (pmol/mg prot.)

50
de 0
xt mM
ro
se
1.5
Pentosidine (pmol/mg protein)
Near infrared
Control
Hyperglycemic
Anaerobic
de 5 m
xt M
ro
se
1
0
x = 315-385nm; m = 400nm x = 325-445nm; m = 460nm x = 325nm; m = 340-500nm
Freshly-harvested porcine dermis
5 week duration
0.5
Significance values
Fluorescence spectra


0
0
Wavelength (nm)
Collagen
features
8
Experimental design

0.5
380
Reflectance

1
10
We undertook this in vitro study to assess any
relationship between multiple optical
spectroscopy modes and the accumulation of
Advanced Glycation Endproducts (AGEs)

1.5
x= 315-385nm;m= 400nm
PCA Factors

Subject studies (see adjacent poster)
demonstrate that NIR & fluorescence can
noninvasively classify diabetes patients with
performance comparable to FPG
NIR transmission mode - absorption spectra
2.5
-log(p)

NIR spectra
Absorption

Prevalence and increasing frequency of
diabetes underscore need for screening
Absorption

0.3
Spectroscopic Prediction (4 PLS Factors)
2
1.5
1
0.5
0
-0.5
0
Number of PLS Factors
10
20
30
40
Time (Experiment Days)
Variance Explained by PLS Model
80

Lysine and CML converted to N-trifluoroacetylmethyl ester derivatives & analyzed by isotope
dilution via selected ion monitoring GC/MS
Pentosidine measured by reverse phase HPLC
using fluorescence detection
*ClinDGInvest,
Dyer et al, Accumulation of Maillard reaction products in skin collagen in diabetes and aging, J
91:2463-9 (1993)
375
FTIR System
Fluorescence: 2 excitation
scans & 1 emission scan
NIR Interferometer
Specimen
Spectral data acquired in
10 sessions over the 5 week
incubation cycle
20
2
15
1.8
10
Control
Hyperglycemic
Anaerobic
Tunable
UV/visible
source
UV/visible
spectrometer
and PMTdetector
400
450
*
500
60
*
40
*
20
0
5
10
20
30
40
x = 325
m = 340 – 500nm
x = 315 - 385
m = 400nm
Near Infrared
x = 325 - 445
m = 460nm
Reflectance
Transmission
The upper figures illustrate representative results from PLS analysis of an excitation fluorescence set (x = 325 – 445 nm; m = 460nm). The upper left figure
illustrates the Standard Error of Prediction as a function of increasing PLS model factors (latent variables). The red circle indicates the optimal number of
model factors. The model predictions of pentosidine concentration vs the study day are plotted in the upper right panel. Similar PLS analysis was performed
for each spectroscopic data set. The variance in pentosidine concentrations explained by the respective models are plotted in the lower figure. The bars
marked by * indicate variance reductions that are statistically significant (p=0.05).
Conclusions
1.6
1.4
Fluorescence
1.2
o
Spectral changes strongly correlated to AGE progression
o
Active spectral regions consistent with collagen cross-linking
0.8
o
Promising method for quantitative in vivo AGE measurement
0.6
NIR
1
0.4
o
Relationship between NIR spectra and AGEs uncertain
0.5
0.2
o
Study results infer AGE chemistry does not have strong NIR signature;
current experiment may lack sufficient sensitivity to identify this
interaction
o
In previous human studies, other diabetes-related skin modifications
may be responsible for classification; necessitates further investigation
2
1.5
0
0
10
20
Study day
Fluorimeter
350
AGE assay results
Pentosidine (pmol/mg protein)
FT-NIR performed in reflectance
and transmission modes
Transmission
425
Fluorescence
Study day
Reflectance

325
Fluorescence spectra were acquired in three wavelength regions/modes. Data from two excitation scans (excitation wavelength varied as detection
wavelength is fixed) are depicted in the left and middle panels. The right panel presents emission scan spectra (excitation wavelength fixed as detection
wavelength is varied). In each of the 3 modes, 4 spectra were acquired from each of 12 specimens at all 10 spectroscopy sessions. Individual traces in the
figures represent the average of those 4 individual spectra. Spectra are color coded to denote in which incubation media they resided.
0
0
NIR Source

375
Wavelength (nm)
Spectroscopy

350
Variance Explained (%)
325
Pentosidine (pmol/mg protein)

CML and pentosidine measured in acid
hydrolysates of collagen & normalized to lysine
(CML) or protein (pentosidine) content
CML (mmol/mol Lysine)

30
40
1
0
0
5
10
15
20
CML (mmol/mol Lysine)
At study outset and weekly thereafter, thin strips (1-2mm in width) of each specimen were excised and frozen (-70°C). At the conclusion study, 48 of the 72
frozen sub-specimens were analyzed for CML and pentosidine content. The assayed samples included all hyperglycemic sub-specimens (24), and one-half
of both the control (12) and anaerobic groups (12). The control and anaerobic subgroups included all of the initial and final sub-specimens from the
respective groups. AGE concentration versus study day are shown in the left-side plots. The pentosidine vs CML concentration for each specimen is plotted
at the right. Clear AGE accumulation occurred in specimens residing in the hyperglycemic solution while minimal accumulation was detected in the control
group. The chelators and anaerobic conditions retarded but did not eliminate AGE accumulation in the specimens contained in that medium. Pentosidine and
CML concentrations in individual specimens are highly correlated. Relative to the hyperglycemic group, however, pentosidine accumulated more rapidly than
CML in the anaerobic and control groups. Each incubation medium thus has a different slope in the right panel.
Acknowledgements
Specimen
This research was funded by LifeScan, Inc.