Impact of diurnal variation, sex, tear collection method, and disease state on tear protein levels in dogs

Abstract

Objective To investigate the effects of various biological factors on total protein concentration (TPC) and serum albumin levels in canine tears. Animals studied 10 healthy beagles (5 female, 5 male) were used. Procedures Experiments were conducted on separate days, collecting tears with either capillary tubes or Schirmer strips, as follows: (i) Tear collection at 3 hours intervals (from 6 am to 12 am); and (ii) Tear collection before and 20 minutes following topical histamine application (1, 10, 375 mg/mL) to induce mild, moderate, and severe conjunctivitis, respectively. TPC and serum albumin were measured with infrared spectroscopy and ELISA, respectively. Results Tear film TPC and serum albumin ranged from 9.7‐26.1 mg/mL and 6.4‐1662.6 µg/mL, respectively. Protein levels did not differ significantly among time points (P ≥ .080). Median coefficient of variation (CV%) was lower with Schirmer strips compared to capillary tubes for both TPC (12% vs 15%, P = .020) and serum albumin (57% vs 78%, P = .232). TPC (P < .001), but not serum albumin was greater in male vs. female dogs. Serum albumin, but not TPC (P ≥ .099), increased significantly with each grade of conjunctivitis severity (P < .001), with no differences between collection devices (P ≥ .322); median increase was 106%, 1389%, and 2871% in eyes with mild, moderate, and severe conjunctivitis, respectively. Conclusions There is no apparent diurnal variation in canine tear protein levels. Blood‐tear barrier breakdown with conjunctivitis allows serum albumin to leak into the tear film at high concentrations. Schirmer strips compare well with capillary tubes for bioanalytical purposes in healthy and diseased eyes, and this collection method may offer improved reproducibility for protein quantification.

Influence of Schirmer strip wetness on volume absorbed, volume recovered, and total protein content in canine tears

“…Ocular disease likely affects the flow rate captured by Schirmer strips (µL/min) due to changes in tear film dynamics (tear volume, turnover rate), but the impact on VA and VR is likely minimal (to absent) as long as the mm‐mark wetness is kept constant (eg, same VA whether 20‐mm is reached within 40 s or 70 s). As for TPC, a recent study showed that Schirmer strips may actually be superior to capillary glass tubes for tear collection in inflamed eyes 5 . In fact, disease state did not exacerbate differences between Schirmer strips and capillary tubes in canine eyes, in contrast to the speculation that Schirmer strips might capture larger protein amounts by causing micro‐trauma to the hyperemic and more vulnerable conjunctiva 5 …”

“…As for TPC, a recent study showed that Schirmer strips may actually be superior to capillary glass tubes for tear collection in inflamed eyes 5 . In fact, disease state did not exacerbate differences between Schirmer strips and capillary tubes in canine eyes, in contrast to the speculation that Schirmer strips might capture larger protein amounts by causing micro‐trauma to the hyperemic and more vulnerable conjunctiva 5 …”

“…Schirmer strips are also increasingly used to sample tear fluid and quantify various components in the canine tear film, including proteins 3 and xenobiotics 4 . Schirmer strips are often considered superior to other collection methods in dogs (eg, capillary tubes, ophthalmic sponges) given improved reproducibility of bioanalytical results 5,6 as well as several other advantages described in a recent review article 7 . For instance, Schirmer strips are well tolerated, easy to use, and yield a relatively large tear sample in a short duration, while capillary tubes require an experienced operator and cooperative patient, and only provide a small volume of tears over a long collection time 7 .…”

Albumin in Tears Modulates Bacterial Susceptibility to Topical Antibiotics in Ophthalmology

“…Accounting for a 2-fold dilution from mixing 1:1 with bacterial broth (see section Antimicrobial Susceptibility Testing), these 10 solutions provided albumin concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.35, 0.5, 0.75, and 1, 2%) that represent a spectrum of albumin detected in tears of healthy canine eyes (generally ≤0.1%) and canine eyes with mild to severe conjunctival inflammation (generally 0.1–2%). This information was based on two previous reports ( 8 , 10 ) as well as prospective evaluation of 25 canine patients diagnosed with ulcerative keratitis at the Iowa State University's Lloyd Veterinary Medical Center, collecting and analyzing tear samples for albumin content as previously described ( 10 ). Briefly, tear collection was performed with dye-free Schirmer strips (Eye Care Product Manufacturing, Tucson, AZ) wetted until 20-mm mark, followed by centrifugation in pre-punctured 0.2-mL tubes at 3,884 g for 2 min and albumin quantification with a canine-specific ELISA kit (Serum albumin ELISA kit, Life Span Biosciences, Inc., Seattle, WA) following the manufacturer's guidelines.…”

“…A large volume of tears (18 mL) was collected from dogs with ocular surface inflammation to provide biological fluid for the subsequent in vitro experiments. First, conjunctivitis was induced in both eyes with topical administration of 1% histamine solution, as previously described ( 8 , 10 ). Twenty minutes were allowed to pass for conjunctivitis to fully develop and ocular surface homeostasis to be restored ( 8 , 15 ).…”