FRUCTATION: CROSS-LINKING OF COLLAGEN HYDROGELS
By
Jackie Lynn Gigante
A capstone project submitted for
Graduation with University Honors
(Date)
University Honors
University of California, Riverside
APPROVED
Dr. Lyubovitsky
Department of Engineering
Dr. Richard Cardullo, Howard H Hays Chair and Faculty Director, University Honors
Associate Vice Provost, Undergraduate Education
ABSTRACT
D-Fructose and proteins can undergo fructation (glycation by fructose) through
the process of a non-enzymatic reaction to create permanently cross-linked fluorescent
derivatives, termed advanced glycation end-products (AGEs). This reaction works
differently for aldoses than for ketoses, which is why a Maillard Reaction with D-fructose
as the reducing sugar gives a Heyn's Product as opposed to the Amadori Product seen in
the Maillard Reaction of D-glucose. Although there is a significant amount of research
that has been done on the D-glucose driven cross-linking and AGE products, there has
been significantly less research examining other sugars as cross-linkers. This research
will aim to further examine fructose as a possible collagen cross-linker.
In order to test the cross-linking effects of fructose, prepared solutions will be
added on top of prepolymerized collagen type I hydrogels. Over the course of 30 days,
one photon fluorescence spectroscopy and multiphoton optical microscopy (MPM)
imaging and spectroscopy will be used to detect the fluorescent structures and to
understand in what ways modification with the sugar alters collagen within the hydrogels.
Fructation and glycation are both significant processes to understand because they
have applications not only to health improvement and age wear reduction, but also to the
increased durability of materials through use of the rigid, heterocyclic products formed
by means of the Maillard Reaction. It is important to consider that different sugars will
cross-link at different rates, creating different and thus not well characterized products.
This research focuses on identifying these key differences using overall fluorescence
intensity, structural imaging, and kinetics. Further research is needed to identify these
AGE end-products, as well as their contributions to the changes in the overall mechanical
properties.
ACKNOWLEDGEMENTS
I would like to take this opportunity to thank my Principal Investigator, Dr. Julia
Lyubovitsky, as well as her graduate student, Xuye Lang, for all the help they provided
and for the opportunity to work in this lab. I would like to thank UC Reagent Research
Start up Funds (JGL), BSAS Grant UC Riverside (JGL), NSF-CBET0847070 (JGL), and
NSF BRIGE EEC-0927297 (JGL) for funding the research in my lab. I would also like to
thank the UCR S.T.EM. Pathway Program, as it was their HSI-STEM Undergraduate
Research Program that helped support me during the course of my research.
TABLE OF CONTENTS
Abstract…………………………………………………………………………………... ii
Acknowledgements……………………………………………………………………….iv
Table of contents…………………………………………………………………………..v
List of Tables and Figures………………………………………………….…………….vi
1. Introduction……………………………………………………………………… 1
2. Experimental………………………………………………………………………
2.1. Fructose………………………………………………………………………
2.2. Multiphoton Imaging Microscopy (MPM).........................................................
2.3 Sucrose…………………………………………………………………….
3. Results………………………………………………………………………………
3.1. DNS Assay………………………………………………………………….
3.2. One Photon Fluorescence Spectroscopy……………………………………..
4. Discussion…………………………………………………………………………
5. References………………………………………………………………………….
List of Reactions
Reaction 1…………………………………………………………………………………
Reaction 2………………………………………………………………………………..
Reaction 3………………………………………………………………………………..
List of Images
Image 1……………………………………………………………………………………
Image 2…………………………………………………………………………………...
List of Figures
Figure 1……………………………………………………………………………………
Figure 2…………………………………………………………………………………...
1. INTRODUCTION
Glycation and fructation are non-enzymatic, posttranslational modifications of
protein. These modifications occur through the Maillard Reaction, which involves a
reaction between a primary amino group of a protein and the carbonyl group of a
reducing sugar molecule (2). This reaction results in the creation of permanently crosslinked, fluorescent products known as advanced glycosylation end-products (AGEs). The
reaction begins with a nucleophilic addition of the protein’s amino group to the carbonyl
group of the sugar (D-fructose). The Schiff Base is then formed through the elimination
of water. Protonation and subsequent deprotonation of the Schiff Base leads to the enol
form of the Heyn's Rearrangement, and finally keto-enol tautomerism leads to the keto
form. This keto product of the Heyn's Rearrangement then reacts with a second molecule
of D-fructose through another nucleophilic addition to the reactive carbonyl group of the
fructose. An intramolecular aldol condensation then leads to the formation of the cyclic
ring. Elimination of two molecules of water gives the substituted pyrrole product
(2,11,15).
The reaction mechanism outlined in Reaction 1 is specific to fructose, but the
process of glycation will also occur with other reducing sugars such as glucose,
galactose, and glyceraldehyde. The substituted pyrrole product noted in the reaction is
one example of a possible AGE, but there are many different possible fluorescent
products that can occur from various side-reactions or with combinations of different
proteins and sugars (some of which can be found in the literature). Although this research
does not focus on the identification of these specific AGEs, from not only fructose but
also other less characterized sugars, this is an area of research that requires further
investigation.
Reaction 1. One possible reaction mechanism for the non-enzymatic fructation reaction of fructose and
collagen, used with permission from SPIE and Ref. 15. A substituted pyrrole is a possible reaction product
for fructation as described in the literature.
Fructose is a monosaccharide sugar, but because it is a ketose (a sugar containing
a ketone group) it is able to exist more readily in the open chain form due to its stability
(7). Although fructose is not absorbed by the body as easily as glucose, studies have
shown that fructose can cross-link with the proteins in the body at 10 times the rate of
glucose (14). Thus, it is important that fructose, and the subsequent AGEs formed from a
Maillard Reaction with this sugar, be better understood as a cross-linker. Fructose is a
component of the human diet in its natural form in honey, dates, berries, etc. Fructose is
also found in the diet as an element of sucrose.
Sucrose, however, is a non-reducing disaccharide and must therefore undergo
inversion before it is able to proceed through the Maillard Reaction. Sucrose can be
hydrolyzed biochemically by invertase, an enzyme found in baker’s yeast (S. cerevisiae)
(1,10). Once sucrose has been hydrolyzed enzymatically into fructose and glucose
(Reaction 2), these products can proceed through the Maillard Reaction to form unique
AGEs. Since sucrose cannot cross-link with collagen prior to hydrolyzation, it is the
effect of the hydrolyzation within the collagen and the simultaneous production of both
glycation and fructation AGEs that is the focus of this particular experiment. In future
directions for this project, it will be imperative to take note of the relative fluorescence
intensity of fructose and collagen cross-linking in the presence of glucose, as the
absorption of fructose is facilitated by the addition of glucose (7). As sucrose is one of
the most common sugars used as sweeteners for human consumption, it is important to
understand the role of sucrose as a cross-linker (4,10).
Reaction 2. Redrawn from Ref. 9. Sucrose is hydrolyzed enzymatically into glucose and fructose
monosaccharides.
AGE accretion occurs over time with normal aging, and has been found in human
cartilage, collagen, and pericardial fluid (13). Accumulation of these AGEs alters the
structural properties of tissue proteins (such as collagen) making them more rigid and
resistant to catabolism (3,11). Studies have also shown that AGE build-up is linked to the
degradation of many other aspects of health, including: bone remodeling and skeletal
fragility, cataract formation, diabetes, and other complications associated with diabetes
(i.e. nephropathy, retinopathy, and neuropathy)(12,13,14). Because of the role they play
in the body, these reactions are imperative to the understanding of possible healthimprovement strategies and reduction of age-induced wear on the body.
Multiphoton microscopy (MPM) is utilized in this experiment because of the
unique properties of this technique. MPM is a non-destructive, high resolution technique
which makes it ideal for in situ testing of delicate biological materials (8). In most
biological tissue, scattering is the optical property that limits depth penetration, as it
leaves only a fraction of the incident light to reach the focus (5,6). However, MPM
utilizes femtosecond pulses of near-infrared (NIR) laser light, which utilizes longer
wavelengths and thus reduces scattering within the non-transparent hydrogels. MPM also
reduces bleaching of fluorophores, which is the photochemical conversion of a
fluorescent species to a non-fluorescent one (5).
MPM combines second harmonic generation (SHG) and two-photon fluorescence
(TPF) contrasts to create images of the cross-linked collagen hydrogels. TPF occurs when
a fluorophore absorbs two photons at a low energy state. Upon absorption, one of its
electrons briefly converts to a higher energy state, and it emits a single, excited photon at
a greater energy level than that of the absorbed photons (8). In SHG, two photons interact
with a non-centrosymmetric target to produce one photon with twice the energy. This
process does not involve absorption of photons, but instead the interaction and scattering
of photons, which does not result in a loss of energy (8,12).
With MPM imaging techniques, an image of the modified collagen fibers can be
produced. From these images, it is possible to visually represent the physical changes
taking place due to sugar modification within the collagen hydrogel. MPM images also
allow for the ability to discern basic information related to the mechanical properties of
the materials such as fiber length and width, as well as effective pore sizes which can be
determined using image analysis multi-platform software programs (e.g. ImageJ, NIH)
(9).
2. EXPERIMENTAL
2.1 FRUCTOSE
In order to test the cross-linking effects of fructose, a collagen hydrogel was
synthesized. The hydrogel was created using Type 1 collagen (4 g/L, pH=7.4) combined
with 2x potassium phosphate buffer. The buffer contained: 0.1929 g/L potassium
phosphate dibasic (K₂HPO₄: Sigma-Aldrich, S7907), 0.1929 g/L potassium phosphate
monobasic (KH₂PO₄: Sigma-Aldrich, S8282), and 1.05192 g/L NaCl (with total buffer
concentration of 0.6 M, pH=7.4) (4). The pH was maintained using dropwise addition of
either HCL or NaOH. The potassium phosphate buffer was then filtered with a 0.22 μm,
25 mm syringe filter (Fisher) and stored at 4 °C. The hydrogel material was formed by
mixing 450 µL (0.6 M) 2x potassium phosphate buffer, 259.1 µL acetic acid, and 380.5
µL Type 1 collagen while maintaining the pH at 7.4 (final collagen concentration = 4
g/L). 110 µL of the collagen hydrogel were then placed into separate wells and covered
with perifilm. For MPM measurements, the collagen hydrogel was prepared in an 8-well
chambers (MP Biomedicals), and for one-photon spectroscopy the collagen hydrogel was
prepared in a 96-well plates (BD Falcon).
The sample was then incubated at 37°C for 24 hours. On top of this hydrogel, a
layer of fructose solution (0.1 M, pH = 7.4) was added. The fructose solution was
prepared by taking 90.08 mg D-fructose (Sigma-Aldrich, F3510) and adding 2x
potassium phosphate buffer to 5 ml. These prepared fructose solutions included 0.112 mL
of an antifungal (Amphotericin B: 22.4 µg/ml) and 0.416 mL of an anti-bacterial
(Gentamicin sulfate: 200 µg/ml; Sigma-Aldrich, G1264) so as to prevent unwanted
growth in the hydrogels that might create anomalous fluorescence signals. The fructose
addition was refreshed regularly as needed.
After the addition of the fructose layer, the hydrogel was then incubated for 35
days at 37°C. At consistent intervals, the hydrogel was removed from incubation in order
to utilize one-photon fluorescence spectroscopy and multiphoton microscopy (MPM)
imaging and spectroscopy to perceive the fluorescent structures and determine in what
ways fructation had altered the form of the collagen. The chambers and/or plates in which
the hydrogel rested were carefully covered in aluminum foil prior to their removal from
the incubation chamber, so as to prevent any photo-bleaching occurring from light
saturation during the transition to the various detection instruments.
2.2 MULTIPHOTON IMAGING MICROSCOPY (MPM)
MPM imaging was taken using a Thorlabs Multiphoton Microscope, which is an
upright multiphoton laser scanning microscope. This laser scanning microscope is
equipped with a titanium:sapphire laser excitation source (Mai-Tai HP, Spectra Physics,
Santa Clara, CA) that provides femtosecond pulses at a repetition rate of about 80 MHz, a
pulse width of <100 femtoseconds, and with the center frequency tunable from 690 to
1040 nm. The two-photon fluorescence (TPF) and second-harmonic generation (SHG)
images found in Image 2A-F were gathered using a long working distance immersion
objective (Zeiss, 63x water, N.A. 1.0). The laser excitation was plane polarized at 810
nm, while dichroic and bandpass filtering (400 – 410 nm, 470 – 550 nm and 570 – 610
nm) were used to isolate the second harmonic (SHG) and induced two-photon
fluorescence (TPF) caused by the fructose modification. Each image presented is 810 ×
650 pixels corresponding to about a 70 μm × 60 μm field of view for each image.
2.3 SUCROSE
Before sucrose could be tested on a collagen hydrogel, the process of sucrose
inversion had to be quantified. Invertase would be used to hydrolyze the sucrose
enzymatically, followed by the DNS assay method to determine what ratio of the sucrose
had been inverted into equimolar amounts of fructose and glucose. In the DNS assay, 3,5dinitrosalicylic acid (Sigma-Aldrich, D0550) is used as the oxidizing agent in an
oxidation-reduction reaction with fructose and/or glucose as the reducing agent (Reaction
3). The reduced product, 3-amino-5-nitrosalicyclic acid, possesses a red-brown color. The
amount of sucrose hydrolyzed can then be determined by taking the absorbance of the
DNS assay samples and comparing the results to a known calibration curve prepared with
known amounts of glucose and/or fructose.
Reaction 3. Glucose or fructose undergo oxidation-reduction reaction with DNS to form 3-amino-5nitrosalicyclic acid which produces the red-brown color used to estimate inversion.
A 10 mg/ml DNS reagent was prepared by combining 400 mg dinitrosalicylic
acid (Sigma-Aldrich, D0550), 20 mg sodium sulfite (Sigma-Aldrich, S0505), and 400 mg
sodium hydroxide (Sigma-Aldrich, 757527) adding double deionized water to 40 ml. The
stock solution of 10 mg/ml glucose in 100 mM sodium acetate buffer to which the DNS
reagent would be added was also prepared. The sodium acetate buffer (100 mM, pH=4.5)
was prepared by mixing 0.41 g sodium acetate (Sigma-Aldrich, S8750) and 50 ml acetic
acid. The pH of the buffer was tested using a Corning 340 pH meter to assure that the pH
level was within ±0.3 of the desired 4.5 operating level for the DNS assay. The 10 mg/ml
glucose solution was prepared by combining 250 mg glucose (Sigma-Aldrich, G7021)
with 25 ml of the 100 mM acetate buffer. A stock solution of 0.4 g/ml potassium tartrate
(Sigma-Aldrich, 60413) was also prepared in double deionized water to stabilize the
color of the DNS assay.
In order to investigate the kinetics of reaction between Glucose/Fructose and
DNS, experiments were designed as shown in the table below (Table 1). Briefly, 0.5 ml
glucose (or 0.25 ml glucose + 0.25 ml fructose)/buffer solution were placed into 2 ml
micro test tubes and 0.5 ml DNS reagent were added. The test tubes were then placed in
a hot water bath at 90℃. Every 2 minutes, a test tube was removed from heating and
allowed to cool down in a room temperature water bath. 0.167 ml of potassium tartrate
were added to stabilize the color. Once all test tubes had cooled down to room
temperature, the mixtures were placed individually into 2 mm light path cuvettes and the
absorbance was taken at 575 nm.
Reaction
Time (min)
0
2
4
6
8
10
11
12
14
DNS (ml)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Glucose
+
Fructose (ml)
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
0.25
+
0.25
Potassium
Tartrate (ml)
0.167 0.167 0.167 0.167
0.167 0.167
Absorbance
at 575 nm
0.01
0.32
0.1
0.22
0.36
0.39
0.167 0.167
-
-
0.167
-
Table 1. Shows the experiment parameters of the DNS assay of glucose and fructose (reaction kinetics).
To prepare a calibration curve, glucose/buffer solutions in varying concentrations
(as shown in Table 2) were placed into 2 ml micro test tubes and 0.5 ml DNS reagent
were added. The test tubes were then placed in a hot water bath at 90 ℃. After six
minutes of incubation in the water bath, 0.167 ml of potassium tartrate were added to
stabilize the color. Once all test tubes had cooled down to room temperature, the
mixtures were placed individually into 1 mm light path cuvettes and the absorbance was
taken at 575 nm. The standard calibration curve of glucose created is shown in Figure 3.
Sodium Acetate Buffer
(ml)
0.5
0.1
0.2
0.3
0.4
Glucose
(ml)
0
0.4
0.3
0.2
0.1
DNS (ml)
0.5
0.5
0.5
0.5
0.5
Potassium Tartrate (ml)
0.167
0.167
0.167
0.167
0.167
Absorbance at 575 nm
0.02
0.33
0.27
0.20
0.11
Table 2. Shows the experiment parameters of the DNS assay of different concentrations of glucose
(calibration curve).
3. RESULTS
3.1. DNS ASSAY
The following figures detail the results utilized to determine the experimental
parameters and procedure for the DNS assay. The results summarized in these graphs
detail how the absorbance wavelength (shown in Figure 1) and the minimum reaction
time (shown in Figure 2) were determined for this experiment. Once these factors had
been determined and the procedure of the DNS assay verified, this experiment was
utilized to create a glucose calibration curve (Figure 3). This calibration curve could then
be used as a reference for future experiments.
Figure 1. Absorbance spectrum for reaction of DNS and glucose. Wavelength range is from 498-700 nm.
Using the typical absorbance spectrum for this reaction (Figure 1), it was
determined that the appropriate wavelength to utilize for the absorbance was 575 nm; at
this wavelength there is a clean, linear slope for the absorbance values of the various
glucose concentrations.
Depending on the reaction time of fructose or glucose with a DNS reagent, a color
gradient from yellow to brown is exhibited (Image 1). This color gradient serves as a
measure for the amount of glucose or fructose that reacts with DNS. By removing the
solutions at specific time intervals and plotting the absorbance versus time, the reaction
rate can be determined (Figure 2).
Image 1. DNS assay for calibrating the rate of reaction between glucose and DNS compound (0.5 ml
glucose/buffer and 0.5 ml DNS incubated at 90℃ for 2, 4, 6, 8, 10, and 12 minutes from left).
Figure 2. Graph of reaction rate of glucose and DNS to produce 3-amino-5-nitrosalicyclic acid colored
reaction product.
A graph of time versus absorbance was created in order to determine the
appropriate incubation time for this experiment. Based on the results of Figure 2, the
reaction rate begins to decline around the 6 minute mark. Thus, 6 minutes was found to
be a sufficient amount of time to incubate the samples in the 90℃ water bath in order to
complete the reaction.
Figure 3. Standard glucose calibration curve was created using different initial glucose concentrations.
The glucose calibration curve created (Figure 3) can then be used as a reference
for the inversion of sucrose. After inverting sucrose with invertase during a future
experiment, it will then be possible to utilize the DNS assay and compare the results to
this standard calibration curve in order to determine what proportion of the sucrose was
converted into glucose and fructose.
3.2. ONE-PHOTON FLUORESCENCE SPECTROSCOPY
In order to detect the fluorescence caused by the AGE’s resulting from fructose
and collagen cross-linking, a FlexStation microplate reader in a backscattering mode
(Molecular Devices) was used. Data was collected using eight different excitations
wavelengths (ranging from 300-405 nm to identify the fluorescence signatures of the
induced fluorescent compounds within the modified collagen hydrogels) and the emission
was collected using the appropriate cutoff filters for these wavelengths. Spectrum was
collected independently from at least two different wells. The spectra were then averaged
and corrected for the background emission by subtracting the background fluorescence.
Measurements were conducted at ~3 day intervals up until 35 days.
The emission peaks resulting from the eight excitation wavelengths used were
graphed as a function of excitation wavelength in Figure 4. Wavelength ranges
appropriate for the cutoff filters used (300 nm and 330 nm having a cutoff filter of 325
nm, which was the best available for this excitation wavelength range; 340-405 nm
having a cutoff filter of 420 nm) were chosen. Results of the one photon fluorescence
revealed that at 330 nm excitation wavelength there was a complimentary emission peak
at 420 nm. For 360 nm excitation wavelength the emission peak fell at 460 nm. Finally,
at 405 nm excitation wavelength, the resulting peak was around 470 nm.
The graph of the one-photon fluorescence data in Figure 4 also shows the relative
intensities of the emissions. Looking at the 340 nm wavelength curve there is a marked
increase in fluorescence intensity on day 31 compared to day 6. This increase in
fluorescence intensity can be attributed to the accumulation of fluorescent AGE products
as fructose continues to cross-link with the collagen over the 35 day span of the
experiment.
Figure 4. One photon fluorescence of fructose modified collagen hydrogels at 6 days (top) and 31 days
(bottom).
From the one photon fluorescence data collected, a general curve of the kinetics
of the fluorescence intensity over time could be computed. Plotting the kinetics data of
fluorescence intensity at 460 nm (Figure 5) revealed the general trend of fluorescence
build-up at that wavelength. Figure 5 shows that there was a general increase of the
fluorescence intensity of the fructose modified collagen hydrogel over the course of 35
days, which can be attributed to the accumulation of fluorescent AGE products. Looking
at the curve, it appears as though there was induction of fluorescence within the first few
days, which is represented by a positive slope. By day 30, the reaction seems to have
fully run its course as there is a decrease in slope and subsequent fluctuation of
fluorescence intensity values.
Figure 5. Kinetics of fructose modification of the collagen hydrogel.
3.3. MULTIPHOTON MICROSCOPY IMAGING
Image 2A-F. MPM Images of 0.1 M fructose-modified collagen hydrogel at Day 4 (Top) and Day 22
(Bottom). Images A and D are SHG images taken through a 400-410 nm filter. Images B and E are TPF
images that were taken through a 470-550 nm fluorescence filter. Images C and F are TPF images that were
taken through a 570-610 nm fluorescence filter. All images were taken with 63X objective and laser power
100 mW.
Looking at the images featured in Image 2, the alteration of the collagen can be
observed from day 4 to day 22 using two-photon fluorescence (TPF) contrast. In the day
4, TPF images (2B and 2C), the collagen fibers are less connected and there does not
appear to be any extended fiber strands. However, by day 22 (Image 2E and 2F) there is a
clear modification of the collagen as the fibers now show connectivity between them (as
highlighted within the colored circles). Fluorescence build-up seems to correlate with
some structural modifications in the materials. The second-harmonic generation (SHG)
images also exhibit collagen modification. At day 4 (Image 2A), the fibers visible in the
SHG image appear small and more separate from each other. However, by day 22 (Image
2D) there is more obvious aggregation visible.
4. DISCUSSION
The higher fluorescent intensity of fructose AGE products versus those of glucose
is most likely due to the higher reactivity of fructose. As a ketose, fructose is able to react
with the amino groups of sugars while in the open-ring form. This means that, unlike
glucose, fructose does not have to undergo cyclization in order to proceed through the
Maillard Reaction, and as such, can cross-link at a much higher rate than glucose.
The next step in looking at sucrose as a collagen cross-linker would be to
determine the experimental parameters for using the invertase to invert sucrose. The
invertase and sucrose concentrations would have to be determined, as well as the rate of
the inversion reaction. Once that has been done, the experiment can proceed as the
fructose experiment did, by adding a layer of inverted sucrose over a collagen hydrogel
and then testing for fluorescence and collagen modification using one-photon
fluorescence and MPM.
Further research is needed to determine how different sugars (especially
disaccharides) will cross-link with the proteins in the human body and what AGE
products will be formed. This information could potentially lead to improvement in the
treatment and management of diet induced diseases, such as diabetes.
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