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Caffeine Increases Tear Volume Depending on Polymorphisms within the Adenosine A2a Receptor Gene and Cytochrome P450 1A2
Caffeine Increases Tear Volume Depending on Polymorphisms within the Adenosine A2a Receptor Gene and Cytochrome P450 1A2 Reiko Arita, MD, PhD,1,2 Yasuo Yanagi, MD, PhD,2 Norihiko Honda, MD, PhD,2 Shuji Maeda, MD, PhD,3 Koshi Maeda, MD, PhD,3 Aya Kuchiba, PhD,4 Takuhiro Yamaguchi, PhD,5 Yoshitsugu Yanagihara, PhD,6 Hiroshi Suzuki, MD, PhD,6 Shiro Amano, MD, PhD2 Purpose: The primary aim of the present study was to examine the effect of caffeine on tear volume. The secondary aim was to investigate the relation between caffeine-induced changes in tear volume and polymorphisms in ADORA2A and CYP1A2. Design: Double-masked, placebo-controlled, crossover study. Participants: Seventy-eight healthy volunteers were recruited for the study. Methods: Subjects participated in 2 sessions in which they received capsules containing either placebo or caffeine. The caffeine capsules were given to the subjects to keep the caffeine volume per body weight within 5 to 7 mg/kg. After caffeine intake, tear meniscus height (TMH) was measured. Subjects provided a blood sample for genotyping. Main Outcome Measures: Tear meniscus height, single nucleotide polymorphism. Results: The tear volume increased after caffeine consumption. The net increase in TMH was 0.08 mm (95% confidence interval, 0.05– 0.10) greater when participants were given caffeine than when given placebo (P⬍0.0001). In ADORA2A, the difference in the net increase in TMH for participants who were heterozygous at rs5751876 and rs2298383 was 0.07 mm (P ⫽ 0.001) and who were minor homozygous was 0.08 mm (P ⫽ 0.007). In CYP1A2, the net increase in TMH for participants who were minor homozygous at rs2472304 was lower than for those who were major homozygous; the difference was 0.06 mm (P ⫽ 0.039). Conclusions: Caffeine intake increases tear volume and polymorphisms within ADORA2A, and CYP1A2 is associated with the tear increase after caffeine intake. Genetic polymorphisms had a significant effect on tear meniscus that was of limited clinical significance. Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. Ophthalmology 2012;119:972–978 © 2012 by the American Academy of Ophthalmology.
Caffeine is the most widely consumed psychoactive substance in the world. Caffeine has various pharmacologic effects in the human body, including relaxation of smooth muscle and stimulation of the central nervous system, cardiac function, and exocrine gland secretion. Because caffeine increases exocrine gland secretion, such as gastric acid and pancreatic secretion,1 caffeine may also increase tear production in the lacrimal glands. To the best of our knowledge, however, the effect of caffeine on tear production or tear volume has not been investigated. The effects of caffeine show greater interindividual variations. Progress has been made in understanding variability in caffeine responses related to the adenosine A2a receptor gene (ADORA2A), cytochrome P450 1A2 (CYP1A2), and, to a more limited extent, dopamine (DRD2)2 and catecholO-methyl transferase (COMT).3 Current research has implicated the caffeine’s main target receptor ADORA2A, and the primary enzyme in caffeine metabolism, CYP1A2, in variability in caffeine response.4 For example, caffeine has
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© 2012 by the American Academy of Ophthalmology Published by Elsevier Inc.
positive effects, such as increased alertness and stimulation, and negative effects, such as increased anxiety. Although the exact mechanisms underlying the interindividual variations in response to caffeine are not clear,5– 8 polymorphisms in the ADORA2A are associated with individual sensitivity to caffeine effects on sleep,9 caffeine-induced anxiety,2,10 and habitual caffeine consumption.11 Because caffeine produces its central effects through its action as an adenosine receptor antagonist,12 the association between caffeine effects and polymorphisms in ADORA2A seems reasonable. However, CYP1A2 accounts for approximately 95% of caffeine metabolism and shows wide interindividual variation in enzyme activity.13 CYP1A2 is associated with neural tube defects,14 myocardial infarction,15 Parkinson disease,16 breast cancer,17 and liver disease.18 Thus, polymorphisms in CYP1A2 might also be associated with the interindividual variations in the response to caffeine. The primary aim of the present study was to examine the effect of caffeine on tear volume. The secondary aim was to ISSN 0161-6420/12/$–see front matter doi:10.1016/j.ophtha.2011.11.033
Arita et al 䡠 Caffeine Increases Tear Volume Depending on Polymorphisms Table 2. Subject Characteristics Variables
Group A
Group B
Total
Gender, n (male/female) Age, years, mean ⫾ (SD) Body weight, kg Mean ⫾ SD Range Caffeine dose per body weight at the test (mg/kg) Wakefulness by caffeine (case number) ⫺ ⫹ Habitual caffeine intake (case number) ⫺ ⫹ mg/d Smoking ⫺ ⫹ Cigarettes/d Schirmer’s value, mean ⫾ SD (mm/5 min, without anesthesia) Tear breakup time, sec (mean ⫾ SD) Tear meniscus height, mm (mean ⫾ SD) Presence of subjective symptoms after caffeine intake (case number) ⫺ ⫹
20/19 35.1⫾11.4
21/18 32.1⫾9.7
41/37 33.6⫾10.6
60.7⫾13.9 40–120 6.2⫾0.5
62.0⫾9.7 40–94 6.3⫾0.5
61.3⫾13.0 40–120 6.3⫾0.5
31 8
27 12
58 20
3 36 427⫾248
2 37 413⫾234
5 73 420⫾240
28 11 18.6⫾9.6 21.5⫾10.2 5.6⫾2.3 0.28⫾0.08
27 12 16.2⫾7.6 20.0⫾10.1 6.0⫾2.3 0.25⫾0.09
55 23 17.3⫾8.5 20.7⫾10.2 5.8⫾2.3 0.26⫾0.09
19 20
24 15
43 35
SD ⫽ standard deviation.
investigate the relation between caffeine-induced changes in tear volume and polymorphisms in ADORA2A and CYP1A2.
Subjects and Methods Subjects Seventy-eight healthy volunteers (41 men and 37 women) were recruited for the study. Subjects were excluded if they had high blood pressure or used any medication on a daily basis. Subjects were also excluded if they had dry eye syndrome, lacrimal drainage obstruction, ocular allergies, ectropion, entropion, trichiasis, open-angle glaucoma, continuous eyedrop use, a history of eye surgery, or ocular diseases that would interfere with tear film production or function. The study protocol was approved by the institutional review board of the University of Tokyo School of Medicine and adhered to the tenets of the Declaration of Helsinki. Before beginning the study, the participants attended a short orientation session in which they read and signed a consent form. This study was conducted between August 2007 and August 2008. This study is registered with University Hospital Medical Information Network in Japan (number UMIN000002903). In our initial analyses, we used only polymerase chain reaction for genetic analyses. However, to be more precise, we added genome sequences to the genotyping. This additional effort, as well as statistical analyses, took approximately 2 years.
Drugs Subjects participated in 2 sessions, separated by ⱖ6 days, in which they received capsules containing either placebo or caffeine. The capsules contained 200 or 300 mg caffeine with a dextrose filler. The placebo capsules contained dextrose alone. All 3 capsules were made at the Department of Pharmacy of the University of
Tokyo. The appearance of the 3 kinds of capsules was indistinguishable. The caffeine capsules were given to subjects in various combinations to keep the caffeine volume per body weight within 5 to 7 mg/kg; 200 mg for body weight less than 41 kg, 300 mg for body weight between 41 and 58 kg, 400 mg for body weight between 58 and 75 kg, 500 mg for body weight between 75 and 93 kg, and 600 mg for body weight ⬎93 kg.
Measurement of Tear Meniscus Height The tear meniscus is present along the superior and inferior lid margins. The tear meniscus is estimated to hold 75% to 90% of the total volume of tears.19 Therefore, careful examination of the lower tear meniscus height (TMH) provides a simple, but clinically useful, indication of the tear volume. To visualize the TMH, a small amount of fluorescein sodium solution (0.5 L of 1% fluorescein sodium solution) was gently dropped into the lower conjunctival sac with a micropipette. After the subject blinked, the center of the lower TMH stained with fluorescein was photographed with a charge-coupled device camera (DXC-C33, SONY, Tokyo, Japan) attached to a slit lamp (SL-7F, TOPCON, Tokyo, Japan) under lighting with a cobaltblue filter (Fig 1, available at http://aaojournal.org). The illumination did not extend to the pupil to prevent reflex tearing. A 1-mm micrometer with 10-m insets (Model OB-M, Olympus, Tokyo, Japan) was photographed under the same magnification and saved as a file on a computer. On a computer display, the distance between the edge of the lower eyelid and the upper edge of the stained tear meniscus was measured as the TMH based on the scale using the 10-m insets. The TMH was measured in the right eye of each subject. To avoid interobserver variability, the tear staining and recording procedures were performed by 1 observer (R.A.). Another observer (S.A.) measured the TMH on the computer display. Both observers were masked with regard to the subjects’ clinical information. To examine the repeatability of the TMH measurement method, 2 sets of measurements were repeated by a single examiner in 10 eyes of 10 normal subjects. The intraexaminer repeatability was
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Ophthalmology Volume 119, Number 5, May 2012 Table 3. Association between the Net Increase in Tear Meniscus Height Heterozygous vs Major Homozygous Gene ADORA2A
CYP1A2
rs Number
Alleles (Major/Minor)
MAF, %‡
Estimate
P Value
rs5751876 rs2298383 rs2236624 rs3743484 rs2472304
T/C C/T C/T G/C G/A
0.45 0.44 0.31 0.20 0.25
0.07 (0.03–0.11) 0.07 (0.03–0.11) 0.01 (⫺0.03–0.05) ⫺0.01 (⫺0.06–0.04) 0.04 (0.00–0.08)
0.001 0.001 0.571 0.654 0.057
Note. Differences and P values were adjusted for time of measurement, gender, age, wakefulness after daily caffeine take, caffeine consumption per day, Estimate is the change in TMH in mm. CI ⫽ confidence interval; MAF ⫽ minor allele frequency.
assessed as the absolute difference of 2 values, |x1 – x2|, where x1 and x2 are the first and second measurements, respectively. The relative amount of variation was evaluated as the percentage of the mean of the 2 measurements, |Nx1 ⫺ x2/[(x1 ⫹ x2)/2] ⫻ 100. To examine the interexaminer reproducibility of the TMH measurement method, 2 examiners took 1 measurement in another 10 eyes of 10 normal subjects, and the interexaminer reproducibility was assessed as the absolute difference of 2 values, |x1 ⫺ x2|, where x1 and x2 are the measurements of the first examiner and second examiners, respectively. The relative amount of variation was evaluated as the percentage of the mean of the 2 measurements, |x1 ⫺ x2/[(x1 ⫹ x2)/2] ⫻ 100.
Sessions This was a double-masked, placebo-controlled, crossover study. Subjects were instructed to abstain from taking any recreational drugs and caffeine for ⱖ6 days before each session. Sessions were conducted from 10 AM to 12 PM with a minimum of 6 days between sessions. The order of administration of placebo or caffeine to each subject was determined by random numbers created with Statistical Analysis System (SAS) software version 9.1.3 (SAS Inc., Cary, NC). Group A comprised 39 participants who were given caffeine first and placebo second. Group B comprised 39 participants who were given placebo first and placebo second. Sessions for women were scheduled without regard to menstrual cycle phase20 or pregnancy. No subjects were allowed to use other prescription or over-the-counter drugs. At 10 AM, TMH was observed and photographed (baseline TMH). The subjects then ingested 1 or 2 capsules with 150 ml of water. At 60 and 120 minutes after capsule consumption, TMH was observed and photographed in the same way. Caffeine is absorbed within 45 minutes of ingestion and the half-life of caffeine is approximately 5.7 hours in healthy adults.21 Moreover, tear stability is at equilibrium between 10 AM and 12 PM.22 Therefore, in the present study, the TMH measurements were set to be performed while the serum caffeine concentration was maintained and tear stability was at equilibrium. The net increase in TMH at 60 minutes was calculated as follows: (net increase in TMH at 60 minutes) ⫽ [(TMH at 60 minutes after caffeine intake) – (TMH before caffeine intake)] – [(TMH at 60 minutes after placebo intake) – (TMH before placebo intake)]. The net increase in TMH at 120 minutes was calculated similarly. On a separate visit, tear film breakup time was measured. Tear function was then evaluated using the Schirmer’s test. Subjects provided a blood sample for genotyping and completed questionnaires regarding age, body weight, caffeine consumption per day, caffeine-induced wakefulness, and smoking habits. Subjects also completed questionnaires to investigate the correlations between responses to caffeine and genotype. After caffeine intake, the
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symptoms related to their conditions (heart rate, blood pressure, urinary trouble) were asked. Pupil size and intraocular pressure were not measured before and after caffeine intake to observe precise TMH because light beam or air pressure might induce the tear film production. Moreover, recent data have suggested that caffeine consumption can raise intraocular pressure in those with open-angle glaucoma, but not in normal controls.23 In the present study, open-angle glaucoma was included in the exclusion criteria.
Genotyping After extracting genomic DNA from the peripheral blood using a kit (DNA Extraction kit, Promega, Madison, WI), the genomic DNA was subjected to direct sequencing. The primer sequences for each single nucleotide polymorphism (SNP) investigated in this study are shown in Table 1 (available at http://aaojournal.org). Based on the allele frequency from the Japanese population, SNPs that were polymorphic within ADORA2A (rs5751876, rs2298383, and rs2236624) and CYP1A2 (rs3743484 and rs2472304) were chosen. Details of the polymerase chain reaction amplification and genotype detection procedures are available upon request. Genotyping was performed with the examiner masked to the subjects’ clinical information. The Hardy–Weinberg equilibrium test was used as a quality control for genotyping; P values were 0.031 (rs5751876), 0.051 (rs2298383), 0.744 (rs2236624), 0.955 (rs3743484), and 0.597 (rs2472304).
Statistical Analysis Power analysis revealed that detection of a 0.1-mm increase in TMH from caffeine intake with a standard deviation of the net increase in TMH of 0.3 in each group and a statistical power of 80% with a 2-tailed ␣ level of 0.05 would require 72 participants (36 participants in each group). Because of repeated measurements of outcomes over time (i.e., measured at 60 and at 120 minutes after capsule intake), we performed a longitudinal data analysis to incorporate all outcome variables. For the primary aim, we compared the increase in TMH from caffeine intake with that from placebo intake using a mixed linear model that included an intercept and time at measurement. For the secondary aim, we analyzed each SNP using a mixed linear model adjusted for time of measurement, gender, age, caffeineinduced wakefulness, daily caffeine intake, smoking habit, and baseline TMH. P values and differences in the net increase in TMH with 95% confidence intervals (CIs) were calculated for each genotype compared with major homozygous and under dominant and recessive models. In this study, the dominant model was defined as “Aa or aa” vs “AA,” where “A” and “a” are major and minor alleles in this study population, respectively, and the recessive model as “aa” vs “AA or Aa.” We also examined other factors that might be associated with the net TMH increase, including gender, age, body weight, dose of
Arita et al 䡠 Caffeine Increases Tear Volume Depending on Polymorphisms Table 3. (TMH) and 5 Single Nucleotide Polymorphisms on 2 Genes Minor Homozygous vs. Major Homozygous
Dominant Model
Recessive Model
Estimate (95% CI), mm
P Value
Estimate (95% CI), mm
P Value
Estimate (95% CI), mm
P Value
0.08 (0.02–0.14) 0.08 (0.02–0.14) ⫺0.05 (⫺0.12–0.01) 0.02 (⫺0.02–0.06) ⫺0.06 (⫺0.11–0.00)
0.007 0.007 0.121 0.346 0.039
0.07 (0.03–0.11) 0.07 (0.03–0.11) 0.00 (⫺0.04–0.04) ⫺0.01 (⫺0.05–0.04) 0.03 (⫺0.01–0.06)
⬍0.001 0.001 0.941 0.739 0.168
0.03 (⫺0.03–0.09) 0.03 (⫺0.03–0.09) ⫺0.06 (⫺0.12–0.00) 0.02 (⫺0.02–0.06) ⫺0.07 (⫺0.12 to –0.02)
0.309 0.309 0.066 0.252 0.008
smoking habit, and baseline TMH.
caffeine given in the current study, caffeine-induced wakefulness, daily caffeine intake, smoking habit, Schirmer’s value, tear film breakup time, and baseline TMH. All these factors, group indicator, and time of measurement were included in the model. P⬍0.05 was considered significant. No adjustment was made for multiple comparisons because of the exploratory nature of the secondary aim. All the statistical analyses were performed using the SAS version 9.1.3.
Results Subject Characteristics All subjects completed both periods of the study. The maximal interval between periods was 6 days. Subject characteristics are shown in Table 2. The mean age of the 78 participants was 33.6 years (range, 20 –54 years), and the mean weight was 61.3 kg (range, 40 –120 kg). Five participants reported no caffeine use, 57 reported consuming between 1 and 5 cups of coffee per day, and 16 reported consuming ⬎6 cups of coffee per day. The mean caffeine consumption per day of the 73 participants that were caffeine users was 4.4 cups (range, 1–10 cups). Habitual caffeine intake per day was calculated based on the daily consumption of coffee and black tea assuming a cup of coffee and black tea contain 100 and 33.8 mg of caffeine, respectively.
Reproducibility of Measuring TMH The interexaminer variability was 0.022⫾0.013 mm (8.77⫾4.84 %) and the intraexaminer variability was 0.007⫾0.007 mm (2.43⫾2.24%) and each variability was small enough.
Effect of Caffeine and Placebo on Tear Volume The net increase in TMH was 0.08 mm (95% CI, 0.05– 0.10) greater when participants were given caffeine than when given placebo (P⬍0.0001). Although an 0.08-mm increase in TMH was slightly less than the definition of clinically significant used in the power calculation, an 0.08-mm increase matches the approximately 30% increase of the pretreatment TMH of 0.26 mm. Thus, an 0.08-mm increase in TMH seems to serve as a clinically significant increase. We asked all subjects about their conditions after caffeine take. A standardized system for the categorization of treatmentemergent adverse events (e.g., Medical Dictionary for Regulatory Activities) was not considered. For the subjective measurements, approximately half of the subjects (35/78) correctly identified caffeine capsules based on subjective symptoms such as heart rate increase, epiphora, perspiration, trembling, palpitations, diuretic
effects, wakefulness, and nasal mucus. The TMH increase at 60 minutes (P ⫽ 0.332, unpaired t test) and 120 minutes (P ⫽ 0.819, unpaired t test) after caffeine administration was not different between subjects with or without subjective symptoms.
Association of SNPs and Other Characteristics of the Participants with the Net Increase in TMH Table 3 shows the differences in the net increase in TMH between genotypes for each SNP. The net increase in TMH of participants who were heterozygous or minor homozygous at rs5751876 was significantly greater than that of participants who were major homozygous. The difference in the net increase in TMH for participants who were heterozygous was 0.07 mm (95% CI, 0.03– 0.11; P ⫽ 0.001), and that for participants who were minor homozygous was 0.08 mm (95% CI, 0.02– 0.14; P ⫽ 0.007). Under the assumption of a dominant model, the difference was 0.07 mm (95% CI, 0.03– 0.11; P⬍0.001). The findings for rs2298383 were similar to those for rs5751876. The D= between rs2298383 and rs5751876 was 0.38, which suggested that these SNPs were not in linkage disequilibrium. The other SNP within ADORA2A, rs2236624, was not significantly associated with the net increase in TMH. In CYP1A2, the net increase in TMH among participants who were minor homozygous at rs2472304 was significantly lower than that among participants who were major homozygous; the difference was 0.06 mm (95% CI, 0.00 – 0.11; P ⫽ 0.039). Under the assumption of a recessive model, the Table 4. Association between the Net Increase in Tear Meniscus Height (THM) and Subject Characteristics Factor Gender (female vs male) Age (per 10 years) Body weight (per 5 kg) Dose of caffeine given in current study (per 100 mg) Wakefulness after daily caffeine intake Daily caffeine intake (per 500 mg) Smoking habit (per 20 cigarettes) Schirmer’s test before examination (per 5 mm) Tear film breakup time before examination (per 5 sec) Baseline TMH (per 0.1 mm)
Estimate (95% CI), mm
P Value
0.02 (⫺0.04–0.08) 0.02 (0.00–0.04) 0.00 (⫺0.01–0.02) 0.01 (⫺0.05–0.06)
0.488 0.021 0.749 0.794
0.08 (0.03–0.12) 0.07 (0.02–0.12) 0.04 (⫺0.01–0.09) 0.00 (⫺0.01–0.01)
0.001 0.006 0.091 0.384
⫺0.03 (⫺0.07–0.01)
0.127
0.03 (0.00–0.06)
0.089
CI ⫽ confidence interval.
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Ophthalmology Volume 119, Number 5, May 2012 difference was 0.07 (95% CI, 0.02– 0.12; P ⫽ 0.008). rs3743484 was not significantly associated with the net increase in TMH. Table 4 shows the association of factors other than SNPs with the net increase in TMH. The net increase in TMH statistically significantly increased with age and daily caffeine intake (0.02 mm [95% CI, 0.00 – 0.04; P ⫽ 0.021] per 10 years of age; 0.07 mm [95% CI, 0.02– 0.12; P ⫽ 0.006] per 5 cups). The net increase in TMH among participants who felt awake after daily caffeine intake was 0.08 mm higher than that among participants who did not feel awake (95% CI, 0.03– 0.12; P ⫽ 0.001). No other factors had a significant effect on TMH.
Discussion The primary finding of the present study was that caffeine significantly increased the tear volume compared with the placebo. Because tear volume is determined by the balance between tear production at the lacrimal glands and removal by the tear drainage system, caffeine administration increased the tear volume by affecting tear production and/or tear drainage. Caffeine increases the production of saliva and gastric acid.1 Moreover, the fluorescein staining of the tears completely disappeared at 60 and 120 minutes after caffeine intake in the present study, suggesting that tear drainage was not decreased by caffeine intake. Thus, it is likely that caffeine increased the tear volume, not by decreasing tear drainage, but by increasing tear production. The secondary finding of this study is that the increase in tear volume after caffeine administration was associated with polymorphisms in ADORA2A and CYP1A2. Although caffeine increased the mean tear volume more than the placebo in the 78 participants, there were interindividual variations in the TMH increase after caffeine administration; TMH increased by ⬎50% in 30 of 78 participants, whereas TMH did not change in most of the remaining participants. Based on previous studies showing variations in the response to caffeine,13,24 –27 we hypothesized that interindividual variations in the tear response after caffeine administration are due to polymorphisms in ADORA2A and/or CYP1A2. Consistent with this hypothesis, we found that the increase in TMH after caffeine administration was associated with polymorphisms within ADORA2A and CYP1A2. The net TMH increases in the subjects with T/C and C/C genotypes at rs5751876 were significantly greater than that in the subjects with T/T. Similarly, the net TMH increases in the subjects with C/T and T/T genotypes at rs2298383 were significantly greater than that in the subjects with C/C. Interestingly, both polymorphisms at rs5751876 and rs2298383 are associated with caffeine-induced anxiety2 and panic disorder.28 These findings might be indicative of a functional variant in rs5751876 and rs2298383. Yu et al29 suggested that the rs2298383 intron 1a polymorphism is located in one of the potential promoter regions upstream of several newly identified ADORA2A exon 1 variants and therefore might be functionally relevant. A previous population-based cohort study found that the prevalence of dry eye is significantly lower in caffeine users (13.0%) than in noncaffeine users (16.6%).30 The authors of the previous study stated that it is not known whether ingested caffeine stimulates tear production. We believe that the present study is the first to show that ingested
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caffeine stimulates tear production. Moreover, the result of the population-based study seems to suggest that caffeine can be a therapeutic drug for dry eye. Even if caffeine becomes a therapeutic drug for dry eye, however, the results of the present study suggest that caffeine should be used selectively in dry eye patients who have a genetic background that is favorable for a caffeine-induced tear volume increase. In the present study, TMH was measured on a computer screen using the image of a scale, which is a novel method. Because the mean (0.26 mm) and the range (0.12– 0.56 mm) of TMH before caffeine intake was similar to those provided in previous reports using various methods,31–35 our method of measuring TMH seems reliable. Schirmer’s test is an invasive method of insertion of a filter paper into the conjunctival sac. Because of the invasiveness, only 1 pair should be done in a given day.36 However, tear film meniscus is a part of the precorneal tear film and results from the reservoir of tear film.37 The TMH is an easily accessible, minimally invasive, indirect measure of the putative function of the lacrimal gland and the patency of the nasolacrimal system. Previous studies showed a positive correlation between TMH and Schirmer’s value in healthy volunteers38 – 40 and dry eye patients.31,41 Because tear volume had to be measured 3 times within 2 hours in this study, TMH was used as a measure of tear volume instead of Schirmer’s test. As shown in the results, the method of measuring TMH has a high reproducibility. This study has some limitations. First, participants were not excluded based on daily caffeine consumption and smoking habit to obtain participants who are representative of the general population. The effect of caffeine tolerance and withdrawal of caffeine, however, might have affected the results of the present study. Moreover, smoking habit may have affected the results because smoking reduces the half-life of caffeine. Second, the present study involved a relatively small number of subjects for a genotype analysis. It is possible that the association between the increase in tear volume and other polymorphisms in ADORA2A and CYP1A2 would have been detected with a larger number of subjects. Third, although one of the hypotheses was a pharmacokinetic hypothesis, we did not measure caffeine pharmacokinetics in this study and the finding on tear meniscus volume might be secondary to other ocular and cardiovascular effects of caffeine. Fourth, although we made conclusions about the mechanism of this caffeine effect, we did not have other measures of tear production, which might lead to calculations of flow, only a tear meniscus measurement. In conclusion, caffeine increases tear volume and the caffeine-induced increase in tear volume is associated with 2 polymorphisms within ADORA2A and CYP1A2.
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Arita et al 䡠 Caffeine Increases Tear Volume Depending on Polymorphisms 3. Happonen P, Voutilainen S, Tuomainen TP, Salonen JT. Catechol-O-methyltransferase gene polymorphism modifies the effect of coffee intake on incidence of acute coronary events. PLoS One [serial online] 2006;1:e117. Available at: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal. pone.0000117. Accessed November 4, 2011. 4. Yang A, Palmer AA, de Wit H. Genetics of caffeine consumption and responses to caffeine. Psychopharmacology (Berl) 2010;211:245–57. 5. Lieberman HR, Wurtman RJ, Emde GG, et al. The effects of low doses of caffeine on human performance and mood. Psychopharmacology (Berl) 1987;92:308 –12. 6. Liguori A, Grass JA, Hughes JR. Subjective effects of caffeine among introverts and extraverts in the morning and evening. Exp Clin Psychopharmacol 1999;7:244 –9. 7. Loke WH. Effects of caffeine on mood and memory. Physiol Behav 1988;44:367–72. 8. Svensson E, Persson LO, Sjoberg L. Mood effects of diazepam and caffeine. Psychopharmacology (Berl) 1980;67:73– 80. 9. Retey JV, Adam M, Khatami R, et al. A genetic variation in the adenosine A2A receptor gene (ADORA2A) contributes to individual sensitivity to caffeine effects on sleep. Clin Pharmacol Ther 2007;81:692– 8. 10. Alsene K, Deckert J, Sand P, de Wit H. Association between A2a receptor gene polymorphisms and caffeine-induced anxiety. Neuropsychopharmacology 2003;28:1694 –702. 11. Cornelis MC, El-Sohemy A, Campos H. Genetic polymorphism of the adenosine A2A receptor is associated with habitual caffeine consumption. Am J Clin Nutr 2007;86:240 – 4. 12. Snyder SH, Sklar P. Behavioral and molecular actions of caffeine: focus on adenosine. J Psychiatr Res 1984;18:91–106. 13. Rasmussen BB, Brix TH, Kyvik KO, Brosen K. The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors. Pharmacogenetics 2002;12:473– 8. 14. Schmidt RJ, Romitti PA, Burns TL, et al. Caffeine, selected metabolic gene variants, and risk for neural tube defects. Birth Defects Res A Clin Mol Teratol 2010;88:560 –9. 15. Cornelis MC, El-Sohemy A, Kabagambe EK, Campos H. Coffee, CYP1A2 genotype, and risk of myocardial infarction. JAMA 2006;295:1135– 41. 16. Tan EK, Chua E, Fook-Chong SM, et al. Association between caffeine intake and risk of Parkinson’s disease among fast and slow metabolizers. Pharmacogenet Genomics 2007;17:1001–5. 17. Jernström H, Henningson M, Johansson U, Olsson H. Coffee intake and CYP1A2*1F genotype predict breast volume in young women: implications for breast cancer. Br J Cancer 2008;99:1534 – 8. 18. Frye RF, Zgheib NK, Matzke GR, et al. Liver disease selectively modulates cytochrome P450-mediated metabolism. Clin Pharmacol Ther 2006;80:235– 45. 19. Holly FJ. Physical chemistry of the normal and disordered tear film. Trans Ophthalmol Soc U K 1985;104:374 – 80. 20. Kamimori GH, Joubert A, Otterstetter R, et al. The effect of the menstrual cycle on the pharmacokinetics of caffeine in normal, healthy eumenorrheic females. Clin Pharmacol Ther 1999;55:445–9. 21. Statland BE, Demas TJ. Serum caffeine half-lives: healthy subjects vs. patients having alcoholic hepatic disease. Am J Clin Pathol 1980;73:390 –3.
22. Patel S, Bevan R, Farrell JC. Diurnal variation in precorneal tear film stability. Am J Optom Physiol Opt 1988;65:151– 4. 23. Li M, Wang M, Guo W, et al. The effect of caffeine on intraocular pressure: a systematic review and meta-analysis. Graefes Arch Clin Exp Ophthalmol 2011;249:435– 42. 24. Castorena-Torres F, Mendoza-Cantu A, de Leon MB, et al. CYP1A2 phenotype and genotype in a population from the Carboniferous Region of Coahuila, Mexico. Toxicol Lett 2005;156:331–9. 25. Chait LD. Factors influencing the subjective response to caffeine. Behav Pharmacol 1992;3:219 –28. 26. Evans SM, Griffiths RR. Dose-related caffeine discrimination in normal volunteers: individual differences in subjective effects and self-reported cues. Behav Pharmacol 1991;2:345–56. 27. Han XM, Ou-Yang DS, Lu PX, et al. Plasma caffeine metabolite ratio (17X/137X) in vivo associated with G-2964A and C734A polymorphisms of human CYP1A2. Pharmacogenetics 2001;11:429 –35. 28. Hohoff C, Mullings EL, Heatherley SV, et al. Adenosine A(2A) receptor gene: evidence for association of risk variants with panic disorder and anxious personality. J Psychiatr Res 2010;44:930 –7. 29. Yu L, Frith MC, Suzuki Y, et al. Characterization of genomic organization of the adenosine A2A receptor gene by molecular and bioinformatics analyses. Brain Res 2004;1000:156 –73. 30. Moss SE, Klein R, Klein BE. Prevalence of and risk factors for dry eye syndrome. Arch Ophthalmol 2000;118:1264 – 8. 31. Kawai M, Yamada M, Kawashima M, et al. Quantitative evaluation of tear meniscus height from fluorescein photographs. Cornea 2007;26:403– 6. 32. Lamberts DW, Foster CS, Perry HD. Schirmer test after topical anesthesia and the tear meniscus height in normal eyes. Arch Ophthalmol 1979;97:1082–5. 33. Lim KJ, Lee JH. Measurement of the tear meniscus height using 0.25% fluorescein sodium. Korean J Ophthalmol 1991; 5:34 – 6. 34. Miller WL, Doughty MJ, Narayanan S, et al. A comparison of tear volume (by tear meniscus height and phenol red thread test) and tear fluid osmolality measures in non-lens wearers and in contact lens wearers. Eye Contact Lens 2004;30:132–7. 35. Tomlinson A, Blades KJ, Pearce EI. What does the phenol red thread test actually measure? Optom Vis Sci 2001;78:142– 6. 36. Lemp MA. Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes. CLAO J 1995;21: 221–32. 37. Mishima S, Gasset A, Klyce SD Jr, Baum JL. Determination of tear volume and tear flow. Invest Ophthalmol 1966; 5:264 –76. 38. Khurana AK, Chaudhary R, Ahluwalia BK, Gupta S. Tear film profile in dry eye. Acta Ophthalmol (Copenh) 1991;69:79 – 86. 39. Savini G, Barboni P, Zanini M. Tear meniscus evaluation by optical coherence tomography. Ophthalmic Surg Lasers Imaging 2006;37:112– 8. 40. Ibrahim OM, Dogru M, Takano Y, et al. Application of Visante optical coherence tomography tear meniscus height measurement in the diagnosis of dry eye disease. Ophthalmology 2010;117:1923–9. 41. Yokoi N, Bron AJ, Tiffany JM, et al. Relationship between tear volume and tear meniscus curvature. Arch Ophthalmol 2004;122:1265–9.
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Ophthalmology Volume 119, Number 5, May 2012
Footnotes and Financial Disclosures Originally received: April 22, 2011. Final revision: November 18, 2011. Accepted: November 30, 2011. Available online: February 14, 2012. 1
5
Division of Biostatistics, Tohoku University Graduate School of Medicine, Miyagi, Japan.
Manuscript no. 2011-631.
Itoh Clinic, Saitama, Japan.
2
Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan.
3
Maeda Ophthalmic Clinic, Fukushima, Japan.
4
Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts.
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6
Department of Pharmacy, University of Tokyo Hospital, Tokyo, Japan.
Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. Correspondence: Reiko Arita, 626-11 Minaminakano, Minuma-ku, Saitama city, Saitama, 337-0042, Japan. E-mail: [email protected].
Caffeine is the most widely consumed psychoactive substance in the world. Caffeine has various pharmacologic effects in the human body, including relaxation of smooth muscle and stimulation of the central nervous system, cardiac function, and exocrine gland secretion. Because caffeine increases exocrine gland secretion, such as gastric acid and pancreatic secretion,1 caffeine may also increase tear production in the lacrimal glands. To the best of our knowledge, however, the effect of caffeine on tear production or tear volume has not been investigated. The effects of caffeine show greater interindividual variations. Progress has been made in understanding variability in caffeine responses related to the adenosine A2a receptor gene (ADORA2A), cytochrome P450 1A2 (CYP1A2), and, to a more limited extent, dopamine (DRD2)2 and catecholO-methyl transferase (COMT).3 Current research has implicated the caffeine’s main target receptor ADORA2A, and the primary enzyme in caffeine metabolism, CYP1A2, in variability in caffeine response.4 For example, caffeine has
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© 2012 by the American Academy of Ophthalmology Published by Elsevier Inc.
positive effects, such as increased alertness and stimulation, and negative effects, such as increased anxiety. Although the exact mechanisms underlying the interindividual variations in response to caffeine are not clear,5– 8 polymorphisms in the ADORA2A are associated with individual sensitivity to caffeine effects on sleep,9 caffeine-induced anxiety,2,10 and habitual caffeine consumption.11 Because caffeine produces its central effects through its action as an adenosine receptor antagonist,12 the association between caffeine effects and polymorphisms in ADORA2A seems reasonable. However, CYP1A2 accounts for approximately 95% of caffeine metabolism and shows wide interindividual variation in enzyme activity.13 CYP1A2 is associated with neural tube defects,14 myocardial infarction,15 Parkinson disease,16 breast cancer,17 and liver disease.18 Thus, polymorphisms in CYP1A2 might also be associated with the interindividual variations in the response to caffeine. The primary aim of the present study was to examine the effect of caffeine on tear volume. The secondary aim was to ISSN 0161-6420/12/$–see front matter doi:10.1016/j.ophtha.2011.11.033
Arita et al 䡠 Caffeine Increases Tear Volume Depending on Polymorphisms Table 2. Subject Characteristics Variables
Group A
Group B
Total
Gender, n (male/female) Age, years, mean ⫾ (SD) Body weight, kg Mean ⫾ SD Range Caffeine dose per body weight at the test (mg/kg) Wakefulness by caffeine (case number) ⫺ ⫹ Habitual caffeine intake (case number) ⫺ ⫹ mg/d Smoking ⫺ ⫹ Cigarettes/d Schirmer’s value, mean ⫾ SD (mm/5 min, without anesthesia) Tear breakup time, sec (mean ⫾ SD) Tear meniscus height, mm (mean ⫾ SD) Presence of subjective symptoms after caffeine intake (case number) ⫺ ⫹
20/19 35.1⫾11.4
21/18 32.1⫾9.7
41/37 33.6⫾10.6
60.7⫾13.9 40–120 6.2⫾0.5
62.0⫾9.7 40–94 6.3⫾0.5
61.3⫾13.0 40–120 6.3⫾0.5
31 8
27 12
58 20
3 36 427⫾248
2 37 413⫾234
5 73 420⫾240
28 11 18.6⫾9.6 21.5⫾10.2 5.6⫾2.3 0.28⫾0.08
27 12 16.2⫾7.6 20.0⫾10.1 6.0⫾2.3 0.25⫾0.09
55 23 17.3⫾8.5 20.7⫾10.2 5.8⫾2.3 0.26⫾0.09
19 20
24 15
43 35
SD ⫽ standard deviation.
investigate the relation between caffeine-induced changes in tear volume and polymorphisms in ADORA2A and CYP1A2.
Subjects and Methods Subjects Seventy-eight healthy volunteers (41 men and 37 women) were recruited for the study. Subjects were excluded if they had high blood pressure or used any medication on a daily basis. Subjects were also excluded if they had dry eye syndrome, lacrimal drainage obstruction, ocular allergies, ectropion, entropion, trichiasis, open-angle glaucoma, continuous eyedrop use, a history of eye surgery, or ocular diseases that would interfere with tear film production or function. The study protocol was approved by the institutional review board of the University of Tokyo School of Medicine and adhered to the tenets of the Declaration of Helsinki. Before beginning the study, the participants attended a short orientation session in which they read and signed a consent form. This study was conducted between August 2007 and August 2008. This study is registered with University Hospital Medical Information Network in Japan (number UMIN000002903). In our initial analyses, we used only polymerase chain reaction for genetic analyses. However, to be more precise, we added genome sequences to the genotyping. This additional effort, as well as statistical analyses, took approximately 2 years.
Drugs Subjects participated in 2 sessions, separated by ⱖ6 days, in which they received capsules containing either placebo or caffeine. The capsules contained 200 or 300 mg caffeine with a dextrose filler. The placebo capsules contained dextrose alone. All 3 capsules were made at the Department of Pharmacy of the University of
Tokyo. The appearance of the 3 kinds of capsules was indistinguishable. The caffeine capsules were given to subjects in various combinations to keep the caffeine volume per body weight within 5 to 7 mg/kg; 200 mg for body weight less than 41 kg, 300 mg for body weight between 41 and 58 kg, 400 mg for body weight between 58 and 75 kg, 500 mg for body weight between 75 and 93 kg, and 600 mg for body weight ⬎93 kg.
Measurement of Tear Meniscus Height The tear meniscus is present along the superior and inferior lid margins. The tear meniscus is estimated to hold 75% to 90% of the total volume of tears.19 Therefore, careful examination of the lower tear meniscus height (TMH) provides a simple, but clinically useful, indication of the tear volume. To visualize the TMH, a small amount of fluorescein sodium solution (0.5 L of 1% fluorescein sodium solution) was gently dropped into the lower conjunctival sac with a micropipette. After the subject blinked, the center of the lower TMH stained with fluorescein was photographed with a charge-coupled device camera (DXC-C33, SONY, Tokyo, Japan) attached to a slit lamp (SL-7F, TOPCON, Tokyo, Japan) under lighting with a cobaltblue filter (Fig 1, available at http://aaojournal.org). The illumination did not extend to the pupil to prevent reflex tearing. A 1-mm micrometer with 10-m insets (Model OB-M, Olympus, Tokyo, Japan) was photographed under the same magnification and saved as a file on a computer. On a computer display, the distance between the edge of the lower eyelid and the upper edge of the stained tear meniscus was measured as the TMH based on the scale using the 10-m insets. The TMH was measured in the right eye of each subject. To avoid interobserver variability, the tear staining and recording procedures were performed by 1 observer (R.A.). Another observer (S.A.) measured the TMH on the computer display. Both observers were masked with regard to the subjects’ clinical information. To examine the repeatability of the TMH measurement method, 2 sets of measurements were repeated by a single examiner in 10 eyes of 10 normal subjects. The intraexaminer repeatability was
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Ophthalmology Volume 119, Number 5, May 2012 Table 3. Association between the Net Increase in Tear Meniscus Height Heterozygous vs Major Homozygous Gene ADORA2A
CYP1A2
rs Number
Alleles (Major/Minor)
MAF, %‡
Estimate
P Value
rs5751876 rs2298383 rs2236624 rs3743484 rs2472304
T/C C/T C/T G/C G/A
0.45 0.44 0.31 0.20 0.25
0.07 (0.03–0.11) 0.07 (0.03–0.11) 0.01 (⫺0.03–0.05) ⫺0.01 (⫺0.06–0.04) 0.04 (0.00–0.08)
0.001 0.001 0.571 0.654 0.057
Note. Differences and P values were adjusted for time of measurement, gender, age, wakefulness after daily caffeine take, caffeine consumption per day, Estimate is the change in TMH in mm. CI ⫽ confidence interval; MAF ⫽ minor allele frequency.
assessed as the absolute difference of 2 values, |x1 – x2|, where x1 and x2 are the first and second measurements, respectively. The relative amount of variation was evaluated as the percentage of the mean of the 2 measurements, |Nx1 ⫺ x2/[(x1 ⫹ x2)/2] ⫻ 100. To examine the interexaminer reproducibility of the TMH measurement method, 2 examiners took 1 measurement in another 10 eyes of 10 normal subjects, and the interexaminer reproducibility was assessed as the absolute difference of 2 values, |x1 ⫺ x2|, where x1 and x2 are the measurements of the first examiner and second examiners, respectively. The relative amount of variation was evaluated as the percentage of the mean of the 2 measurements, |x1 ⫺ x2/[(x1 ⫹ x2)/2] ⫻ 100.
Sessions This was a double-masked, placebo-controlled, crossover study. Subjects were instructed to abstain from taking any recreational drugs and caffeine for ⱖ6 days before each session. Sessions were conducted from 10 AM to 12 PM with a minimum of 6 days between sessions. The order of administration of placebo or caffeine to each subject was determined by random numbers created with Statistical Analysis System (SAS) software version 9.1.3 (SAS Inc., Cary, NC). Group A comprised 39 participants who were given caffeine first and placebo second. Group B comprised 39 participants who were given placebo first and placebo second. Sessions for women were scheduled without regard to menstrual cycle phase20 or pregnancy. No subjects were allowed to use other prescription or over-the-counter drugs. At 10 AM, TMH was observed and photographed (baseline TMH). The subjects then ingested 1 or 2 capsules with 150 ml of water. At 60 and 120 minutes after capsule consumption, TMH was observed and photographed in the same way. Caffeine is absorbed within 45 minutes of ingestion and the half-life of caffeine is approximately 5.7 hours in healthy adults.21 Moreover, tear stability is at equilibrium between 10 AM and 12 PM.22 Therefore, in the present study, the TMH measurements were set to be performed while the serum caffeine concentration was maintained and tear stability was at equilibrium. The net increase in TMH at 60 minutes was calculated as follows: (net increase in TMH at 60 minutes) ⫽ [(TMH at 60 minutes after caffeine intake) – (TMH before caffeine intake)] – [(TMH at 60 minutes after placebo intake) – (TMH before placebo intake)]. The net increase in TMH at 120 minutes was calculated similarly. On a separate visit, tear film breakup time was measured. Tear function was then evaluated using the Schirmer’s test. Subjects provided a blood sample for genotyping and completed questionnaires regarding age, body weight, caffeine consumption per day, caffeine-induced wakefulness, and smoking habits. Subjects also completed questionnaires to investigate the correlations between responses to caffeine and genotype. After caffeine intake, the
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symptoms related to their conditions (heart rate, blood pressure, urinary trouble) were asked. Pupil size and intraocular pressure were not measured before and after caffeine intake to observe precise TMH because light beam or air pressure might induce the tear film production. Moreover, recent data have suggested that caffeine consumption can raise intraocular pressure in those with open-angle glaucoma, but not in normal controls.23 In the present study, open-angle glaucoma was included in the exclusion criteria.
Genotyping After extracting genomic DNA from the peripheral blood using a kit (DNA Extraction kit, Promega, Madison, WI), the genomic DNA was subjected to direct sequencing. The primer sequences for each single nucleotide polymorphism (SNP) investigated in this study are shown in Table 1 (available at http://aaojournal.org). Based on the allele frequency from the Japanese population, SNPs that were polymorphic within ADORA2A (rs5751876, rs2298383, and rs2236624) and CYP1A2 (rs3743484 and rs2472304) were chosen. Details of the polymerase chain reaction amplification and genotype detection procedures are available upon request. Genotyping was performed with the examiner masked to the subjects’ clinical information. The Hardy–Weinberg equilibrium test was used as a quality control for genotyping; P values were 0.031 (rs5751876), 0.051 (rs2298383), 0.744 (rs2236624), 0.955 (rs3743484), and 0.597 (rs2472304).
Statistical Analysis Power analysis revealed that detection of a 0.1-mm increase in TMH from caffeine intake with a standard deviation of the net increase in TMH of 0.3 in each group and a statistical power of 80% with a 2-tailed ␣ level of 0.05 would require 72 participants (36 participants in each group). Because of repeated measurements of outcomes over time (i.e., measured at 60 and at 120 minutes after capsule intake), we performed a longitudinal data analysis to incorporate all outcome variables. For the primary aim, we compared the increase in TMH from caffeine intake with that from placebo intake using a mixed linear model that included an intercept and time at measurement. For the secondary aim, we analyzed each SNP using a mixed linear model adjusted for time of measurement, gender, age, caffeineinduced wakefulness, daily caffeine intake, smoking habit, and baseline TMH. P values and differences in the net increase in TMH with 95% confidence intervals (CIs) were calculated for each genotype compared with major homozygous and under dominant and recessive models. In this study, the dominant model was defined as “Aa or aa” vs “AA,” where “A” and “a” are major and minor alleles in this study population, respectively, and the recessive model as “aa” vs “AA or Aa.” We also examined other factors that might be associated with the net TMH increase, including gender, age, body weight, dose of
Arita et al 䡠 Caffeine Increases Tear Volume Depending on Polymorphisms Table 3. (TMH) and 5 Single Nucleotide Polymorphisms on 2 Genes Minor Homozygous vs. Major Homozygous
Dominant Model
Recessive Model
Estimate (95% CI), mm
P Value
Estimate (95% CI), mm
P Value
Estimate (95% CI), mm
P Value
0.08 (0.02–0.14) 0.08 (0.02–0.14) ⫺0.05 (⫺0.12–0.01) 0.02 (⫺0.02–0.06) ⫺0.06 (⫺0.11–0.00)
0.007 0.007 0.121 0.346 0.039
0.07 (0.03–0.11) 0.07 (0.03–0.11) 0.00 (⫺0.04–0.04) ⫺0.01 (⫺0.05–0.04) 0.03 (⫺0.01–0.06)
⬍0.001 0.001 0.941 0.739 0.168
0.03 (⫺0.03–0.09) 0.03 (⫺0.03–0.09) ⫺0.06 (⫺0.12–0.00) 0.02 (⫺0.02–0.06) ⫺0.07 (⫺0.12 to –0.02)
0.309 0.309 0.066 0.252 0.008
smoking habit, and baseline TMH.
caffeine given in the current study, caffeine-induced wakefulness, daily caffeine intake, smoking habit, Schirmer’s value, tear film breakup time, and baseline TMH. All these factors, group indicator, and time of measurement were included in the model. P⬍0.05 was considered significant. No adjustment was made for multiple comparisons because of the exploratory nature of the secondary aim. All the statistical analyses were performed using the SAS version 9.1.3.
Results Subject Characteristics All subjects completed both periods of the study. The maximal interval between periods was 6 days. Subject characteristics are shown in Table 2. The mean age of the 78 participants was 33.6 years (range, 20 –54 years), and the mean weight was 61.3 kg (range, 40 –120 kg). Five participants reported no caffeine use, 57 reported consuming between 1 and 5 cups of coffee per day, and 16 reported consuming ⬎6 cups of coffee per day. The mean caffeine consumption per day of the 73 participants that were caffeine users was 4.4 cups (range, 1–10 cups). Habitual caffeine intake per day was calculated based on the daily consumption of coffee and black tea assuming a cup of coffee and black tea contain 100 and 33.8 mg of caffeine, respectively.
Reproducibility of Measuring TMH The interexaminer variability was 0.022⫾0.013 mm (8.77⫾4.84 %) and the intraexaminer variability was 0.007⫾0.007 mm (2.43⫾2.24%) and each variability was small enough.
Effect of Caffeine and Placebo on Tear Volume The net increase in TMH was 0.08 mm (95% CI, 0.05– 0.10) greater when participants were given caffeine than when given placebo (P⬍0.0001). Although an 0.08-mm increase in TMH was slightly less than the definition of clinically significant used in the power calculation, an 0.08-mm increase matches the approximately 30% increase of the pretreatment TMH of 0.26 mm. Thus, an 0.08-mm increase in TMH seems to serve as a clinically significant increase. We asked all subjects about their conditions after caffeine take. A standardized system for the categorization of treatmentemergent adverse events (e.g., Medical Dictionary for Regulatory Activities) was not considered. For the subjective measurements, approximately half of the subjects (35/78) correctly identified caffeine capsules based on subjective symptoms such as heart rate increase, epiphora, perspiration, trembling, palpitations, diuretic
effects, wakefulness, and nasal mucus. The TMH increase at 60 minutes (P ⫽ 0.332, unpaired t test) and 120 minutes (P ⫽ 0.819, unpaired t test) after caffeine administration was not different between subjects with or without subjective symptoms.
Association of SNPs and Other Characteristics of the Participants with the Net Increase in TMH Table 3 shows the differences in the net increase in TMH between genotypes for each SNP. The net increase in TMH of participants who were heterozygous or minor homozygous at rs5751876 was significantly greater than that of participants who were major homozygous. The difference in the net increase in TMH for participants who were heterozygous was 0.07 mm (95% CI, 0.03– 0.11; P ⫽ 0.001), and that for participants who were minor homozygous was 0.08 mm (95% CI, 0.02– 0.14; P ⫽ 0.007). Under the assumption of a dominant model, the difference was 0.07 mm (95% CI, 0.03– 0.11; P⬍0.001). The findings for rs2298383 were similar to those for rs5751876. The D= between rs2298383 and rs5751876 was 0.38, which suggested that these SNPs were not in linkage disequilibrium. The other SNP within ADORA2A, rs2236624, was not significantly associated with the net increase in TMH. In CYP1A2, the net increase in TMH among participants who were minor homozygous at rs2472304 was significantly lower than that among participants who were major homozygous; the difference was 0.06 mm (95% CI, 0.00 – 0.11; P ⫽ 0.039). Under the assumption of a recessive model, the Table 4. Association between the Net Increase in Tear Meniscus Height (THM) and Subject Characteristics Factor Gender (female vs male) Age (per 10 years) Body weight (per 5 kg) Dose of caffeine given in current study (per 100 mg) Wakefulness after daily caffeine intake Daily caffeine intake (per 500 mg) Smoking habit (per 20 cigarettes) Schirmer’s test before examination (per 5 mm) Tear film breakup time before examination (per 5 sec) Baseline TMH (per 0.1 mm)
Estimate (95% CI), mm
P Value
0.02 (⫺0.04–0.08) 0.02 (0.00–0.04) 0.00 (⫺0.01–0.02) 0.01 (⫺0.05–0.06)
0.488 0.021 0.749 0.794
0.08 (0.03–0.12) 0.07 (0.02–0.12) 0.04 (⫺0.01–0.09) 0.00 (⫺0.01–0.01)
0.001 0.006 0.091 0.384
⫺0.03 (⫺0.07–0.01)
0.127
0.03 (0.00–0.06)
0.089
CI ⫽ confidence interval.
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Ophthalmology Volume 119, Number 5, May 2012 difference was 0.07 (95% CI, 0.02– 0.12; P ⫽ 0.008). rs3743484 was not significantly associated with the net increase in TMH. Table 4 shows the association of factors other than SNPs with the net increase in TMH. The net increase in TMH statistically significantly increased with age and daily caffeine intake (0.02 mm [95% CI, 0.00 – 0.04; P ⫽ 0.021] per 10 years of age; 0.07 mm [95% CI, 0.02– 0.12; P ⫽ 0.006] per 5 cups). The net increase in TMH among participants who felt awake after daily caffeine intake was 0.08 mm higher than that among participants who did not feel awake (95% CI, 0.03– 0.12; P ⫽ 0.001). No other factors had a significant effect on TMH.
Discussion The primary finding of the present study was that caffeine significantly increased the tear volume compared with the placebo. Because tear volume is determined by the balance between tear production at the lacrimal glands and removal by the tear drainage system, caffeine administration increased the tear volume by affecting tear production and/or tear drainage. Caffeine increases the production of saliva and gastric acid.1 Moreover, the fluorescein staining of the tears completely disappeared at 60 and 120 minutes after caffeine intake in the present study, suggesting that tear drainage was not decreased by caffeine intake. Thus, it is likely that caffeine increased the tear volume, not by decreasing tear drainage, but by increasing tear production. The secondary finding of this study is that the increase in tear volume after caffeine administration was associated with polymorphisms in ADORA2A and CYP1A2. Although caffeine increased the mean tear volume more than the placebo in the 78 participants, there were interindividual variations in the TMH increase after caffeine administration; TMH increased by ⬎50% in 30 of 78 participants, whereas TMH did not change in most of the remaining participants. Based on previous studies showing variations in the response to caffeine,13,24 –27 we hypothesized that interindividual variations in the tear response after caffeine administration are due to polymorphisms in ADORA2A and/or CYP1A2. Consistent with this hypothesis, we found that the increase in TMH after caffeine administration was associated with polymorphisms within ADORA2A and CYP1A2. The net TMH increases in the subjects with T/C and C/C genotypes at rs5751876 were significantly greater than that in the subjects with T/T. Similarly, the net TMH increases in the subjects with C/T and T/T genotypes at rs2298383 were significantly greater than that in the subjects with C/C. Interestingly, both polymorphisms at rs5751876 and rs2298383 are associated with caffeine-induced anxiety2 and panic disorder.28 These findings might be indicative of a functional variant in rs5751876 and rs2298383. Yu et al29 suggested that the rs2298383 intron 1a polymorphism is located in one of the potential promoter regions upstream of several newly identified ADORA2A exon 1 variants and therefore might be functionally relevant. A previous population-based cohort study found that the prevalence of dry eye is significantly lower in caffeine users (13.0%) than in noncaffeine users (16.6%).30 The authors of the previous study stated that it is not known whether ingested caffeine stimulates tear production. We believe that the present study is the first to show that ingested
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caffeine stimulates tear production. Moreover, the result of the population-based study seems to suggest that caffeine can be a therapeutic drug for dry eye. Even if caffeine becomes a therapeutic drug for dry eye, however, the results of the present study suggest that caffeine should be used selectively in dry eye patients who have a genetic background that is favorable for a caffeine-induced tear volume increase. In the present study, TMH was measured on a computer screen using the image of a scale, which is a novel method. Because the mean (0.26 mm) and the range (0.12– 0.56 mm) of TMH before caffeine intake was similar to those provided in previous reports using various methods,31–35 our method of measuring TMH seems reliable. Schirmer’s test is an invasive method of insertion of a filter paper into the conjunctival sac. Because of the invasiveness, only 1 pair should be done in a given day.36 However, tear film meniscus is a part of the precorneal tear film and results from the reservoir of tear film.37 The TMH is an easily accessible, minimally invasive, indirect measure of the putative function of the lacrimal gland and the patency of the nasolacrimal system. Previous studies showed a positive correlation between TMH and Schirmer’s value in healthy volunteers38 – 40 and dry eye patients.31,41 Because tear volume had to be measured 3 times within 2 hours in this study, TMH was used as a measure of tear volume instead of Schirmer’s test. As shown in the results, the method of measuring TMH has a high reproducibility. This study has some limitations. First, participants were not excluded based on daily caffeine consumption and smoking habit to obtain participants who are representative of the general population. The effect of caffeine tolerance and withdrawal of caffeine, however, might have affected the results of the present study. Moreover, smoking habit may have affected the results because smoking reduces the half-life of caffeine. Second, the present study involved a relatively small number of subjects for a genotype analysis. It is possible that the association between the increase in tear volume and other polymorphisms in ADORA2A and CYP1A2 would have been detected with a larger number of subjects. Third, although one of the hypotheses was a pharmacokinetic hypothesis, we did not measure caffeine pharmacokinetics in this study and the finding on tear meniscus volume might be secondary to other ocular and cardiovascular effects of caffeine. Fourth, although we made conclusions about the mechanism of this caffeine effect, we did not have other measures of tear production, which might lead to calculations of flow, only a tear meniscus measurement. In conclusion, caffeine increases tear volume and the caffeine-induced increase in tear volume is associated with 2 polymorphisms within ADORA2A and CYP1A2.
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Ophthalmology Volume 119, Number 5, May 2012
Footnotes and Financial Disclosures Originally received: April 22, 2011. Final revision: November 18, 2011. Accepted: November 30, 2011. Available online: February 14, 2012. 1
5
Division of Biostatistics, Tohoku University Graduate School of Medicine, Miyagi, Japan.
Manuscript no. 2011-631.
Itoh Clinic, Saitama, Japan.
2
Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan.
3
Maeda Ophthalmic Clinic, Fukushima, Japan.
4
Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts.
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6
Department of Pharmacy, University of Tokyo Hospital, Tokyo, Japan.
Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. Correspondence: Reiko Arita, 626-11 Minaminakano, Minuma-ku, Saitama city, Saitama, 337-0042, Japan. E-mail: [email protected].