Article Date: 11/1/2005

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, PhD

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.

IMPROVED PROTEOMICS

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
profile 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 lens wearers.

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. CLS

Creating Protein Profiles

Proteomics 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 electrophoresis separation.

Initially, 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 Two-Dimensional Electrophoresis
Time Fast throughput  Time consuming
Reproducibility Good Poor
Sensitivity High Low


Contact Lens Spectrum, Issue: November 2005