11_04 CLS_AMO SUPPLEMENT
Measuring Tear Film Integrity
Can lens care solutions change the protein
composition of the tear film in contact lens wearers? New analytic technology is
helping answer this question.
By Franz H. Grus, MD,
Proteins and peptides
in the tear film help preserve the integrity and stability of the ocular surface
by mediating inflammatory processes and regulating epithelial growth.1
Ocular surface disease,2,3 systemic disease,4,5 environmental
factors6,7 and contact lens wear can change the protein profile of the
tear film and make the ocular surface more susceptible to inflammation and infection.
Recently, my colleagues and I used a new proteomics
system to compare the tear film compositions of contact lens wearers and non-contact
lens wearers. We also used this technology to measure tear film protein profile
changes in contact lens wearers who switched to a different multipurpose solution
for the duration of the study.
The Surface Enhanced Laser
Desorption/Ionization (SELDI) ProteinChip spectrometer uses ProteinChip arrays to
capture, identify and profile protein patterns.
A biological sample, such as tear fluid,
is placed onto a chromatic chip surface. The different selective surfaces of the
chromatic chip cation or anion exchange surfaces, hydrophobic surfaces and
metal-binding surfaces bind different proteins. Surface-enhanced laser desorption
ionization separates the proteins from the sample and classifies them according
to their physical properties. Each protein is graphed to show the protein
of the biological sample.
We used SELDI ProteinChip technology
to examine protein and peptide expression in the tear fluid of healthy subjects
and patients with dry-eye syndrome.1 This system is particularly sensitive
to the low-molecular mass proteins found in tears. See "Creating Protein Profiles"
for descriptions of other proteomics testing methods.
The SELDI system correctly differentiated
between the eye fluid of
patients with dry eyes (n = 88) and patients with healthy
eyes (n = 71) with a sensitivity and specificity of approximately 90%.1
In patients with dry eyes, we found that some of the potentially protective biomarkers
were downregulated and some inflammatory markers were upregulated. For example,
the proline-rich proteins that are thought to mediate modulation of surface microflora
were significantly downregulated in the dry eye group.
Satisfied with the sensitivity
and reproducibility of SELDI ProteinChip technology, we used this device to compare
low-molecular weight proteins in the tears of contact lens wearers and non-contact
PROTEIN PROFILES AND
LENS CARE SOLUTIONS
Our pilot study comprised
29 healthy, non-contact lens wearers (control group) and 25 contact lens wearers.
Patients in the contact lens group continued wearing their customary lenses to exclude
the influence of the lenses on the protein profiles. However, they did switch from
their usual lens care solution to Complete MoisturePlus multipurpose solution for
the duration of the 4-week study.8
We used SELDI ProteinChip technology
to evaluate tear film protein profiles of patients in both groups. The control group
was tested three times during the study and the contact-lens group was tested six
times: Once before switching lens care products and 2 days, 1 week, 2 weeks, 3 weeks
and 4 weeks after switching to Complete MoisturePlus. Baseline examination included
a survey of subjective symptoms, such as burning, itching, foreign body sensation,
dryness and photophobia and Schirmer tests with anesthesia.
We prepared tears collected from Schirmer
tests for ProteinChip analysis by eluting samples from the first 10 mm of the test
strips overnight. Our previous dry eye study confirmed that no protein was lost
during elution1 and that Schirmer strip tear film extracts were comparable
to those from standard microcapillary tear samples.
When the Schirmer elutions were placed
on the ProteinChip arrays, the system detected more than 370 proteins and peptides
over the groups and clustered the results for further analysis. The most important
biomarkers for our study were purified and identified by mass spectrometry.
Schirmer testing and patients' subjective
responses were not sensitive enough to differentiate between subjects who wore contact
lenses and those who didn't. However, the ProteinChip system showed that the patients
in the contact lens group had statistically significant differences in tear composition
from patients in the control group. The system differentiated between contact lens
wearers and non-contact lens wearers with 90% specificity and 95% sensitivity.
On subsequent evaluations, we noted
changes in the tear film protein profiles of contact lens patients. After using
Complete MoisturePlus multipurpose solution for 2 weeks, the contact lens wearers'
tear profiles were no longer statistically significantly different from those of
the non-contact lens wearers. By the end of the study, most of the contact lens
wearers' tear protein profiles were similar to the typical tear protein profiles
seen in non-contact lens wearers.
Initially, the proline-rich proteins
in contact lens wearers' tear films were downregulated, much like the tears of dry
eye patients in our previous study. These same proteins upregulated to normal values
as patients continued to use Complete MoisturePlus multipurpose solution during
the study. These results suggest that lens care solutions may play an important
role in improving tear protein profiles in contact lens wearers.
TEAR FILM MAINTENANCE
At the end of the 4-week
study, we concluded that contact lenses interact with and alter tear protein
profiles in a complex manner, and contact lens care solutions can influence
these effects. Our study suggests Complete MoisturePlus multipurpose solution does
not disrupt tear film proteins and helps patients maintain a contact lens–tear
film environment similar to that of non-contact lens wearers.
We also concluded that tear
protein analysis might be a noninvasive way to search for objective biomarkers and
identify changes in tear protein profiles with other market multipurpose solutions.
Creating Protein Profiles
is an analytic method that identifies proteins involved in disease processes and
determines how they're up- or downregulated relative to normal, non-disease states.
My colleagues and I use this method to profile proteins in tear fluids.
Available proteomics methods for analyzing tear
fluids include high-performance liquid chromatography (HPLC), two-dimensional electrophoresis
and SELDI ProteinChip technology.
High-performance liquid chromatography
separates molecules under high pressure in a solid-matrix filled stainless steel
column. Next, mass spectrometry and electrochemical detectors identify the separated
molecules and create a protein profile for the analyzed sample.
Two-dimensional electrophoresis is
a more specific proteomics test for tear film proteins than other standard clinical
tests, such as the Schirmer (basal secretory) test and tear breakup time.9
This method can detect differences between the tear film protein profiles of patients
with dry eye syndrome and those of healthy subjects.10 However, two-dimensional
electrophoresis is time-consuming, difficult to reproduce and inadequately sensitive
to low-molecular weight proteins smaller than 20 kDA.
In contrast, SELDI ProteinChip technology
is sensitive to peptides as small as 200 Da and easily analyzes the small sample
volume of tear fluids. This technology's accuracy, reproducibility and ability to
identify small-mass proteins measures tear film protein profiles better than conventional
the proline-rich proteins of contact lens wearers' tears were downregulated, much
like the tears of dry eye patients in our previous study.
Comparison of Proteomics Techniques
||SELDI ProteinChip Technology
Contact Lens Spectrum, Issue: November 2005