Clinical Trial of Docosahexaenoic Acid in Patients with Retinitis Pigmentosa Receiving Vitamin a Treatment

Home , Docosahexaenoic acid

CLINICAL SCIENCES Clinical Trial of Docosahexaenoic Acid in Patients With Retinitis Pigmentosa Receiving Vitamin A Treatment

Eliot L. Berson, MD; Bernard Rosner, PhD; Michael A. Sandberg, PhD; Carol Weigel-DiFranco, MA; Ann Moser, BA; Robert J. Brockhurst, MD; K. C. Hayes, DVM, PhD; Chris A. Johnson, PhD; Ellen J. Anderson, RD; Alexander R. Gaudio, MD; Walter C. Willett, MD; Ernst J. Schaefer, MD

Objective: To determine whether a therapeutic dose of acid plus vitamin A (DHA+A) group and control plus docosahexaenoic acid (DHA), an ␻-3 fatty acid, will slow vitamin A (control+A) group over a 4-year interval the course of retinal degeneration in adult patients with among all 221 randomized patients or among the 208 retinitis pigmentosa who are also receiving vitamin A. patients who completed all 4 follow-up visits. The mean annual rate of loss of sensitivity for the Hum- Design: Randomized, controlled, double-masked trial phrey Field Analyzer 30-2 program was 37 dB for the of 221 patients, aged 18 to 55 years, evaluated over a 4-year DHA+A group and 38 dB for the control+A group interval. Patients were given either 1200 mg/d of doco- (P=.88). For the Humphrey Field Analyzer 30-2 and sahexaenoic acid or control capsules. All were given 15000 30/60-1 programs combined, the mean annual rates of IU/d of vitamin A (given as retinyl palmitate). Random- loss of field sensitivity were 57 dB for the DHA+A ization considered genetic type and baseline dietary ␻-3 group and 60 dB (P=.73) for control+A group. No fatty acid intake. toxic adverse effects were observed. No significant differences by treatment group assignment were observed Main Outcome Measures: The primary outcome mea- within genetic types or within the category of baseline sure was the total point score for the 30-2 program of ␻-3 fatty acid intake. the Humphrey field analyzer; secondary outcome measures were the total point score for the 30-2 and 30/60-1 Conclusion: In patients assigned to receive 15000 IU/d programs combined, 30-Hz electroretinogram ampli- of vitamin A, this randomized trial showed that 1200 mg/d tude, and Early Treatment Diabetic Rentinopathy Study of docosahexaenoic acid supplementation over a 4-year visual acuity. interval did not, on average, slow the course of disease in patients with retinitis pigmentosa. Results: No significant differences in decline in ocular function were found between the docosahexaenoic Arch Ophthalmol. 2004;122:1297-1305

ETINITIS PIGMENTOSA HAS A We and others have found that some worldwide prevalence of patients with retinitis pigmentosa have de- about 1 in 4000 persons.1-10 creased mean plasma and red blood cell Affected patients typically (RBC) docosahexaenoic acid (DHA) con- show elevated dark-adapta- centrations.18-24 Rod outer segment mem- tionR thresholds,11,12 constricted visual fields, branes contain exceptionally large amounts retinal arteriolar attenuation, intraretinal of polyunsaturated fatty acids, especially pigment around the midperiphery, and re- DHA. In fact, they compose almost half of duced and delayed electroretinograms the esterified fatty acids in the outer segment phospholipids.25-27 Docosahexae- See also page 1306 noic acid is a long chain ␻-3 fatty acid found particularly in some fish (eg, salmon (ERGs).13,14 Histological studies have shown and tuna). The RBC DHA levels have been that visual loss occurs owing to degenera- strongly correlated with DHA levels in the tion of rod and cone photoreceptors.15,16 A retina (r=0.88).28 The proportion of DHA prior randomized, controlled trial showed in outer segment phosphatidylethanol- that patients treated with vitamin A palmi- amine (PE) relative to other fatty acids is tate, 15000 IU/d, had, on average, a slower typically 10- to 15-fold higher than in non- rate of decline of retinal function as moni- neural tissue such as RBC membranes.25,27 Author affiliations are listed at tored by the ERG compared with those not The special lipid composition of rod outer the end of this article. receiving this dose.17 segments is thought to be necessary to

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1297

©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 able for analysis at year 3 or 4 of follow-up (n=61), we Table 1. Eligibility Criteria found that the decline in 30-Hz ERG amplitude in these patients over a 4-year interval was inversely related to Ocular criteria the RBC PE DHA concentration (P=.03) for all genetic Retinitis pigmentosa, typical forms* types combined. These results persisted after control- Best-corrected visual acuity Ն20/100 HFA 30-2 program total point score Ն250 dB† ling for age, sex, genetic type, baseline ERG amplitude, 30-Hz cone ERG amplitude Ն0.68 µV and treatment group assignment in a multiple regres- No confounding ocular disease‡ sion model (P=.05). Dietary criteria The significant positive relationship between RBC Dark fish intake Յ5 servings per week§ PE DHA concentration and the preservation of ERG am- ␻ Յ ࿣ Dietary -3 fatty acid intake 0.41 g/d plitude, the known role of DHA in maintaining normal No intake of cod liver oil or ␻-3 capsules Preformed vitamin A (retinol) intake in diet and supplements photoreceptor function, and the evidence that vitamin Յ10 000 IU/d A slows the average decline in ERG amplitude in pa- Supplement intake Յ5000 IU/d of vitamin A and Յ30 IU/d of vitamin E tients with retinitis pigmentosa prompted us to conduct Consumption Յ3 alcoholic beverages per day this randomized, controlled clinical trial to determine Medical and other criteria whether orally administered docosahexaenoic acid can Age range, 18-55 y halt or slow the course of the typical forms of retinitis BMI Ͻ40 and weight Ն5th percentile for age, sex, and height Serum retinol level Յ100 µg/dL and serum retinyl ester levels pigmentosa among patients receiving vitamin A (given Յ380 nmol/L as retinyl palmitate) therapy. Serum cholesterol level Ͻ300 mg/dL and serum triglyceride levels Ͻ400 mg/dL No clinically significant abnormal result on CBC or serum liver function profile METHODS Not pregnant or planning to become pregnant Agree not to know study capsule content PROTOCOL No other disease that might affect absorption or metabolism of docosahexaenoic acid or vitamin A We first conducted a pilot study on 35 patients with the typical forms of retinitis pigmentosa who received 600 mg/d (n=9) Abbreviations: BMI, body mass index (calculated as weight in kilograms of docosahexaenoic acid, 1200 mg/d (n=9) of docosahexae- divided by the square of height in meters); CBC, complete blood cell count; noic acid, or a placebo control (n=17) over 3 months to de- ERG, electroretinogram; HFA, Humphrey Field Analyzer. termine to what extent these dosages raised the concentration SI conversion factors: To convert cholesterol to millimoles per liter, of RBC PE DHA in the short-term. All participants were also multiply by 0.0259; triglycerides to millimoles per liter, multiply by 0.01129. treated with 15000 IU/d of vitamin A. Both dosages of doco- *Elevated final dark-adaptation threshold, retinal arteriolar narrowing, and reduced and delayed ERGs; patients with atypical forms such as paravenous sahexaenoic acid at least doubled the RBC PE DHA concentra- retinitis pigmentosa (RP), sector RP, unilateral RP, Refsum disease, tion and were safely tolerated in this population. We chose the Bardet-Biedl syndrome, and retinitis punctata albescens were excluded. higher dosage of docosahexaenoic acid for this trial assuming Patients who had RP with profound congenital deafness were also excluded. that any therapeutic effect would be more readily detected with †A size V test light was used. the higher dosage. ‡Includes glaucoma, uveitis, diabetic retinopathy, posterior subcapsular We screened patients with retinitis pigmentosa for eligi- cataract greater than 11% of the total lens area (ie, equivalent to P3 on the Lens Opacities Classification System III), and pupil diameter after dilation bility according to ocular, dietary, and medical criteria smaller than 6 mm. (Table 1). We performed a baseline examination on eligible §Dark fish was defined as salmon, mackerel, bluefish, or swordfish. patients within 8 weeks of the screening examination. At base- ࿣Estimated from reported intake of tuna fish, dark fish, light fish, and shell line, patients were randomly assigned to 1 of 2 groups—those fish. who received a supplement of 6 capsules per day each containing 500 mg of fatty acids of which 200 mg was docosahexa- maintain cellular membrane disc fluidity for the normal enoic acid for a total of 1200 mg/d of docosahexaenoic acid or functioning of rhodopsin as it changes conformation in those who received 6 placebo capsules per day containing 500 the initial stages of phototransduction.26,29-33 Further- mg of fatty acids with no docosahexaenoic acid. The docosa- more, it has been proposed that a concentration gradi- hexaenoic acid–enriched capsules, provided by Martek Biosci- ent of DHA normally exists in the subretinal space be- ences Corporation, Columbia, Md, contained a vegetable oil from microalgae with ascorbyl palmitate (Ͻ0.001 mg) and to- tween the rod outer segments (higher concentration) and copherols (Ͻ0.001 mg) as antioxidants. The fatty acid com- the retinal pigment epithelium (lower concentration) and position was as follows: capric, 0 to 10 mg; lauric, 0 to 30 mg; that the release of 11-cis retinal from interphotorecep- myristic, 50 to 100 mg; palmitic, 50 to 100 mg; palmitoleic, 0 tor retinoid-binding protein is facilitated when interpho- to 10 mg; stearic, 0 to 10 mg; oleic, 50 to 150 mg; linoleic, 0 to toreceptor retinoid-binding protein is exposed to a suf- 25 mg; docosahexaenoic, 190 to 210 mg; nervonic, 0 to 10 mg; ficient DHA concentration in the subretinal space.34-37 and others, 0 to 15 mg. The control capsules, also provided by Because RBC membrane phospholipid levels are simi- Martek Biosciences Corporation, contained a mixture of 50% corn and 50% soybean oil with ascorbyl palmitate (Ͻ0.001 mg) lar in composition to, and change in parallel with, reti- Ͻ nal phospholipid levels with dietary manipulation,38-41 we and tocopherols ( 0.001 mg) as antioxidants. Their fatty acid evaluated the DHA content of RBC PE in patients with composition was as follows: palmitic, 45 to 55 mg; stearic, 10 to 20 mg; oleic, 110 to 125 mg; and ␣-linolenic, 15 to 25 mg. retinitis pigmentosa. We found that RBC PE DHA levels Patients were instructed to take 3 capsules in the morning and in our patients were significantly lower, on average, than 3 capsules in the evening. Both the docosahexaenoic acid plus 24 in subjects with normal vision. In an analysis of longi- vitamin A (DHA+A)–supplemented group and the control plus tudinal data from participants in our previous trial of vi- vitamin A (control+A) group received3goffatty acids from tamin A and/or vitamin E for whom we had RBCs avail- these capsules; on a 2000-calorie diet this constituted 1.4% of

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1298

Follow-up Year

Variable Screening Baseline 1234 Medical history Yes Yes Yes Yes Yes Yes Dietary intake Yes No Yes Yes Yes Yes Ophthalmological findings Yes Yes Yes Yes Yes Yes Standard laboratory tests* Yes No Yes Yes Yes Yes Serum retinol and retinyl ester levels Yes No Yes Yes Yes Yes Plasma DHA level Yes Yes Yes Yes Yes Yes RBC PE DHA level No Yes Yes No No Yes Compliance† No No Yes Yes Yes Yes Family history form Yes No No No No No

Abbreviations: DHA, docosahexaenoic acid; PE, phosphatidylethanolamine; RBC, red blood cells. *These included the following: complete blood cell count, liver function profile, serum cholesterol (ie, total, high-density lipoprotein, low-density lipoprotein) level, and serum triglyceride levels. †Completed at each visit and at 6-month intervals between visits by telephone after 6-month supply of capsules was mailed.

calories as fat or an increase of 27 cal/d. All patients were given and Safety Monitoring Committee needed to consider all in- vitamin A as 15000 IU of retinyl palmitate in tablets (Akorn formation from several measures of efficacy and safety in mak- Ophthalmics, Buffalo Grove, Ill) and instructed to take 1 tab- ing informed decisions about stopping the study. let daily with breakfast. Patients completed a food frequency questionnaire42,43 and medical questionnaire at each visit with ASSIGNMENT the aid of a clinical coordinator; they were followed up annually over 4 years (Table 2). Plasma and RBC PE DHA levels The procedure for randomization considered the estimated di- were monitored as measures of compliance,44 and serum reti- etary intake and genetic type at the screening examination. A nol and retinyl ester levels as well as serum liver function test method of block randomization was used within 2 groups of results were evaluated to help exclude any toxic effects of vi- ␻-3 fatty acid intake (above and below the median of 0.16 g/d tamin A therapy.45 determined from a food frequency questionnaire adminis- We used the measurement of static perimetric sensitivi- tered to patients in our previous therapeutic trial of vitamin ties (ie, total point score) with the 30-2 program size V target A) and 6 genetic types (dominant without known mutation, in the Humphrey Field Analyzer (HFA) (Carl Zeiss Ophthal- dominant with known rhodopsin or rhodopsin/peripherin mu- mic Systems, Inc, Dublin, Calif) as the primary outcome mea- tation, recessive, X-linked, isolate, or undetermined [eg, sure. The size V target was used to minimize the number of adopted]) or 12 strata. Within each stratum, patients were ran- locations with floor effects (sensitivity Յ0 dB). The FASTPAC domized in equal numbers to the DHA+A group and control+A test strategy was used to test both central (30-2) and periph- group in blocks of size 4. A separate set of randomization as- eral (30/60-1) visual fields in as short a time as possible.46-48 signments was maintained for each stratum based on a computer- Because the area of the visual field is statistically related to ERG generated set of random numbers that was available only to a amplitude49 and because our preliminary data derived from the programmer who provided assignment information to the data 30-Hz ERG amplitude, we used the full-field 30-Hz ERG am- manager (C.W.D.) on a case-by-case basis. Group assignment plitude as a secondary outcome measure. Visual acuity (Early was implemented by the data manager. Treatment Diabetic Retinopathy Study [ETDRS])50 and the total point score to a size V target with the HFA 30-2 and 30/60-1 MASKING programs combined were also followed up as secondary outcome measures. All members of the staff in contact with the patients, includ- We estimated that 220 patients were needed to provide ing the principal investigator (E.L.B.), were masked with sufficient power (ie, ␣=.05, ␤=.10) to observe a statistically regard to each patient’s treatment group assignment. Each significant difference (29 dB) between mean change in the ocular examination was performed without review of previous DHA+A and control+A groups with respect to HFA 30-2 total records. All serum samples were analyzed without knowledge point score over a 4-year interval and allowing for 5% attri- of treatment group assignment. Patients did not know the tion. The project was approved by the institutional review boards contents of the supplement under study or their treatment of the Massachusetts Eye and Ear Infirmary and Harvard Medi- group assignment and also agreed not to know the course of cal School, Boston, and the study conformed to the Declara- their retinal degeneration until the end of the study. Treat- tion of Helsinki. All patients signed an informed consent form ment group assignments and plasma DHA and RBC PE DHA prior to the screening examination and, if eligible, prior to the levels were placed in records separate from that used for ocu- baseline examination as well. The trial was funded by the Na- lar examinations as part of masking those in contact with the tional Eye Institute as a single-center study. A Data and Safety patients. Monitoring Committee selected by the National Eye Institute met with the investigators prior to the onset of the study to ap- DATA ANALYSIS prove the protocol and annually thereafter in closed session to review the results for both patient safety and efficacy. The study Outcome data for a given patient for each visit represented the was planned to allow 4 years of follow-up for each patient. The average of test results from both eyes or for a single eye if data Lan-DeMets ␣-spending approach with an O’Brien-Fleming for the other eye were unavailable. Visual field data (total point boundary with 5 looks was prespecified51 as the stopping rule. scores) were analyzed separately for the central field (HFA 30-2 However, this stopping rule was just a guideline as the Data program) and for total field (HFA 30-2 and 30/60-1 programs

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1299

©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Table 3. Baseline Demographic and Ocular Characteristics of 208 Patients With Retinitis Pigmentosa*

Patients Given DHA + A Patients Given Control + A Variable (n = 105) (n = 103) P Value† Genetic type, No. (%) of patients Dominant 20 (19) 19 (18) Dominant with mutation 6 (6) 4 (4) Recessive 16 (15) 13 (13) .92 X-linked 5 (5) 8 (8) Isolate 50 (48) 50 (49) Undetermined 8 (8) 9 (9) Dietary intake ␻-3 Fatty acids, g/d 0.19 ± 0.01 0.18 ± 0.01 .71 Multivitamin use, No. (%) who answered yes 23 (22) 22 (21) .99 Blood values‡ RBC PE DHA, % of total RBC PE fatty acids 4.77 ± 0.18 4.27 ± 0.14 .03 Plasma DHA, % of total plasma fatty acids 1.65 ± 0.07 1.51 ± 0.06 .12 Serum retinol level, µg/dL 55.4 ± 1.30 53.9 ± 1.30 .42 Serum retinyl ester levels, nmol/L 140.0 ± 5.70 134.8 ± 5.60 .51 Sex, No. (%) male 50 (48) 56 (54) .40 Age, y 37.8 ± 0.90 36.0 ± 1.00 .19 Weight, kg Male 82.3 ± 1.80 82.5 ± 1.70 .95 Female 64.1 ± 1.60 65.5 ± 2.10 .59 Baseline ocular function§ HFA total point scores, dB HFA 30-2 field 806.4 ± 45.20 912.4 ± 47.40 .11 HFA total field 1203 ± 69 1309 ± 72 .29 ERG amplitudes, µV࿣ 30 Hz 1.34 ± 0.11 (3.82) 1.26 ± 0.11 (3.53) .60 0.5 Hz 2.97 ± 0.12 (19.49) 3.10 ± 0.12 (22.20) .46 ETDRS visual acuity, No. of letters 51.3 ± 1.00 51.4 ± 0.90 .99

Abbreviations: control + A, control capsules plus vitamin A (given as retinyl palmitate); DHA + A, docosahexaenoic acid plus vitamin A; ERG, eletroretinogram; ETDRS, Early Treatment Diabetic Retinopathy Study; HFA, Humphrey Field Analyzer; PE, phosphatidylethanolamine; RBC, red blood cell. *Patients were randomized to 1 of 2 treatment groups with follow-up visits at all 4 years. Patients received either 1200 mg/d of docosahexaenoic acid plus 15 000 IU/d of vitamin A (DHA + A) or control capsules plus 15 000 IU/d of vitamin A (control + A). Data are given as mean ± SE unless otherwise indicated. †P value for t test comparing 2 groups for continuous variables: P value for ␹2 for categorical variables. ‡Lower normal level for RBC PE DHA level equals 3.16%; for plasma DHA level, 0.86%. The reference ranges for the levels are as follows: serum retinol, 38 to 100 µg/dL; serum retinyl ester, 34 to 380 nmol/L. Sample sizes for the DHA + A and control + A groups, respectively, were RBC PE DHA, 104 and 100 patients; plasma DHA, 103 and 100; serum retinol, 105 and 103; and retinyl esters, 105 and 103. §Lower normal level for HFA 30-2 program field (size V target) was 2500 dB; HFA total field (size V target), 4200 dB. The lower normal level for 30-Hz ERG was 50 µV; lower normal level for 0.5-Hz ERG, 350 µV. The lower normal level for ETDRS visual acuity was 60 letters; 51 letters indicated a Snellen visual acuity of 20/30; sample sizes for DHA + A and control + A groups, respectively, were: HFA 30-2 program, 103 and 102 patients; HFA total field, 102 and 102; 30-Hz ERG, 101 and 100; 0.5-Hz ERG, 61 and 53; and EDTRS visual acuity, 105 and 103.

࿣Values designated as mean loge ± SE. Geometric means for ERG amplitudes are given in parentheses.

combined) when both were available. If an eye became pseu- equals 0, 1, 2, ...,4;eit, error term that represents within- dophakic after the baseline visit, visual field data for that eye person variation for the ith patient at time t; and Corr (eit1,eit2) were analyzed only for those visits prior to cataract surgery. If =r. We used PROC MIXED to fit model 1 where both a and the total point score for a visual field became zero, the visit at b=(b1, b2, b3) are assumed to be random. which the zero score was first obtained was included in the analy- Although the baseline value is not explicitly specified as ses but subsequent visits were censored. Slopes and changes a covariate in equation 1, it is implicitly adjusted for because from baseline were computed for each patient, and average slopes of the correlation between the error terms for the baseline visit and mean changes were evaluated for each parameter of ocu- and each follow-up visit. In particular, the marginal model in lar function by treatment group. Comparisons by assigned treat- equation 1 can be rewritten in conditional form as follows: ment group were also performed within genetic type and within category (ie, above and below the intake of 0.16 g/d) at base- (2) Yit=[a(1−R)]+RYi0+[b1(1−R)Gi]+b2t+b3tGi+uit, line of dietary ␻-3 fatty acid intake. Longitudinal regression where Corr(u ,u ) equals [R/(1+R)]. analyses were then performed based on PROC MIXED of SAS it1 it2 Equation 2 is a conditional model. Thus, b can be inter- version 6.12.52 2 preted as the slope in the control+A group (ie, group 2), b +b The regression models that we used in this clinical trial 2 3 can be interpreted as the slope in the DHA+A group (ie, group generally have the following form: 1), and b3 is the difference in slope between the DHA+A group and the control+A group after controling for baseline values.53 (1) Y =a+b G +b t+b tG +e , it 1 i 2 3 i it Two hundred eight patients were seen for all 4 years of where Yit indicates outcome variable (eg, HFA 30-2 total point follow-up and the results will focus on these patients. Sensi- score) for the ith subject at time t; Gi, indicator variable for treat- tivity analyses were performed on all 221 patients randomized ment group, equals 1 if patient i is in group 1, equals 0 if pa- (1) for all available data and (2) after using multiple imputa- tient i is in group 2; t, time in years from the screening visit, t tion methods to account for missing data.54

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1300

18 PARTICIPANT FLOW AND FOLLOW-UP 16

From May 13, 1996, to September 26, 1997, we examined 14 456 patients from across the United States to identify 221 patients (1 per family) with retinitis pigmentosa who met 12 the preset list of eligibility criteria. Two hundred eight of 10 these patients completed all 4 annual follow-up visits. Base- line characteristics of these 208 patients for factors used 8 in the stratification of the randomization (ie, genetic type 6 and dietary ␻-3 fatty acid intake) are given in Table 3 by 4 assigned treatment group. Also given is baseline information concerning the distribution of demographic factors, of RBC PE Fatty Acids RBC PE DHA, % of Total 2

ocular function, and laboratory measures of DHA and reti- 0 nol concentrations. Ninety-seven percent of the eligible pa- Baseline Follow-up Baseline Follow-up tients had intraretinal bone spicule pigmentation around DHA + A Group Control + A Group the midperipheral fundus. Eleven percent of patients reported partial hearing loss. Six percent of the study popu- Figure 1. Red blood cell phosphatidylethanolamine docosahexaenoic acid (RBC PE DHA) levels as a percentage of total RBC PE fatty acids in patients lation were minorities. With the exception of RBC PE DHA with retinitis pigmentosa supplemented with docosahexaenoic acid plus levels, no significant differences in any of the above pa- vitamin A (vitamin A given as retinyl palmitate) (DHA+A) or control plus tient characteristics were noted between the DHA+A and vitamin A (control+A) measured prior to randomization (baseline) or as the mean of measurements at 1 year and 4 years subsequent to randomization control+A groups. Fifty percent of patients in the DHA+A (follow-up) among all patients in the 208 patient cohort for whom group and 64% of patients in the control+A group had cata- measurements were available. Sample sizes were 104 and 100 at baseline racts in at least 1 eye at baseline (P=.05). and 102 and 100 at follow-up for the DHA+A and control+A groups, respectively. Values have been displaced horizontally to facilitate visualization. SAFETY AND COMPLIANCE

Capsule counts indicated that 92% of the docosahexa- Prior to treatment, the mean±SE RBC PE DHA per- enoic acid capsules, 92% of the control capsules, and 94% centage of total RBC PE fatty acids was significantly higher of the vitamin A tablets were consumed over all 4 years. in the DHA+A group (4.77%±0.18%) vs the control+A Similar results were seen with returned monthly calen- group (4.27%±0.14%) (P=.03) (Table 3). At year 1 of fol- dars. One patient in the control+A group diagnosed as low-up the mean±SE RBC PE DHA percentages of total RBC having breast cancer just before her year 4 visit died 4 PE fatty acids for the DHA+A group vs the control+A group months after the year 4 visit. No other patient reported were 12.62%±0.28% and 4.90%±0.18%; at year 4 the mean the development of cancer to us during the study. No pa- RBC PE DHA percentages of total fatty acids were tient experienced a complete loss of vision in an eye over 12.83%±0.05% and 4.66%±0.18% for the 2 groups, re- the course of the study. Furthermore, there was no evi- spectively. Averaging years 1 and 4, the mean±SE change dence of systemic illness or a toxic effect that was attrib- from baseline for the DHA+A group (7.95±0.30) and mean utable to the study capsules or to vitamin A during the change from baseline for the control+A group (0.51±0.11) course of this study based on complete blood cell counts, were both significantly different from zero (PϽ.001) and liver function assessments, patient responses to a symp- were significantly different from each other (PϽ.001). The tom questionnaire, and on the serum retinol and serum RBC PE DHA values ranged from 2% to 18% of the total retinyl ester levels. Two patients were examined by the RBC PE fatty acids prior to treatment in both groups and consulting internist, 1 for gastrointestinal distress thought at follow-up from 3% to 17% of the total RBC PE fatty ac- subsequently to be due to anxiety and the other for dis- ids in the DHA+A group and from 2% to 10% in the coloration of the skin that proved subsequently to be re- control+A group (Figure 1). lated to Cushing syndrome. Table 4 lists serum triglyceride and cholesterol lev- Prior to treatment, the DHA+A and control+A els at screening and year 4 for the 2 groups. The differ- groups showed comparable levels of plasma DHA (Table ence in slopes for the 2 groups was significant for the lev- 3). At follow-up, mean±SE plasma DHA percentages els of triglycerides, total cholesterol, and low-density (mean of all follow-up measurements) of total plasma fatty lipoprotein cholesterol. acids were 5.12%±0.12% and 1.61%±0.07% for the DHA+A group vs the control+A group, respectively; mean ANALYSIS OF OUTCOME MEASURES change from baseline for the DHA+A group was significantly different from the mean change from baseline for Figure 2 shows mean±SE values by year and treat- the control+A group (PϽ.001). By year 1 the mean plasma ment group for central field (HFA 30-2 program) sensi- DHA percentage of total fatty acids was 3-fold higher in tivity, total field (HFA 30-2 and 30/60-1 programs com- the DHA+A group than in the control+A group and re- bined) sensitivity, 30-Hz ERG amplitude, and ETDRS mained at that level for the 4 years of follow-up; no sig- visual acuity. Similar declines can be seen in all out- nificant increase in plasma DHA percentage was noted come measures in the DHA+A group vs the control+A in the control+A group. group. Mean change analyses from time zero (mean of

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1301

Follow-up

Variable Screening Year 4 Slope P Value† Serum triglyceride levels, mg/dL‡ DHA + A group§ 94.5 ± 5.8 93.6 ± 5.9 −0.90 Ͻ.001 Control + A group࿣ 92.9 ± 5.8 123.3 ± 10.6 4.52 Serum total cholesterol level, mg/dL DHA + A group 188.8 ± 3.8 199.9 ± 3.2 2.25 .02 Control + A group 186.0 ± 4.1 188.3 ± 3.6 0.25 Serum high-density lipoprotein cholesterol level, mg/dL DHA + A group 48.0 ± 1.3 58.5 ± 1.5 2.72 .97 Control + A group 45.7 ± 1.1 55.3 ± 1.7 2.74 Serum low-density lipoprotein cholesterol, mg/dL DHA + A group 122.1 ± 3.3 123.1 ± 3.3 −0.54 Ͻ.001 Control + A group 121.6 ± 3.5 110.8 ± 3.0 −3.21

Abbreviations: control + A, control capsules plus vitamin A (vitamin A given as retinyl palmitate); DHA + A, docosahexaenoic acid plus vitamin A. SI conversion factors: To convert triglycerides to millimoles per liter, multiply by 0.01129; all cholesterol levels to millimoles per liter, multiply by 0.0259. *Data are given as the mean ± SE unless otherwise indicated. †The P value is based on slopes. ‡Analyses for triglyceride levels were performed on log values. §Patients received 1200 mg/d of docosahexaenoic acid plus 15 000 IU/d of vitamin A. ࿣Patients received control capsules plus 15 000 IU/d of vitamin A.

Control + A Group DHA + A Group

1000 1400

950 1350 1300 900 1250 850 1200 800 1150

750 1100 1050 700 1000 650 950 Mean Total Visual Field Sensitivity, dB Visual Field Sensitivity, Mean Total Mean Central Visual Field Sensitivity, dB Mean Central Visual Field Sensitivity, 600 900 01234 01234 Year of Follow-up Year of Follow-up

1.5 53

1.4 52 v µ

e 1.3 51 1.2

1.1 50

1.0 49 0.9 Mean ETDRS Acuity, Letters Mean ETDRS Acuity, Mean ERG Amplitude,log 48 0.8

0.7 47 01234 01234 Year of Follow-up Year of Follow-up

Figure 2. The mean central visual field sensitivity (Humphrey Field Analyzer [HFA] 30-2 program, size V target), the mean total visual field sensitivity (HFA 30-2 and 30/60-1 programs combined, size V target), the mean full-field, 30-Hz ERG amplitude, and the mean Early Treatment Diabetic Retinopathy Study visual acuity by year and treatment assignment (control plus vitamin A [vitamin A given as retinyl palmitate] [control+A] group vs docosahexaenoic acid plus vitamin A [DHA+A] group) for patients seen at all 4 years of follow-up. Symbols and error bars designate mean±SE, respectively. Numbers of patients for the control+A group and the DHA+A group, respectively, are 102 and 103 (upper left panel), 102 and 102 (upper right panel), 100 and 101 (lower left panel), and 102 and 104 (lower right panel).

screening and baseline values) did not reveal statisti- Table 5 summarizes the mean annual rates of de- cally significant differences between the 2 treatment cline of visual field sensitivity to the HFA 30-2 program groups for any of these 4 measures. and the HFA 30-2 and 30/60-1 programs combined, of

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1302

©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Table 5. Annual Rate of Decline Over 4 Years for Measures of Ocular Function by Treatment Group*

Patients Given DHA + A Patients Given Control + A Variable (n = 105)† (n = 103)† P Value‡ HFA 30-2 program field, dB/y 36.95 ± 3.36‡ (103) 37.68 ± 3.36 (102) .88 HFA total field, dB/y§ 57.21 ± 4.90 (102) 59.59 ± 4.90 (102) .73

30-Hz ERG amplitude, loge 0.10 ± 0.01 (101) 0.11 ± 0.01 (100) .64 % decline per year࿣ 9.92 10.49 ETDRS visual acuity, No. of letters per year 0.71 ± 0.12 (104) 0.68 ± 0.12 (102) .86

Abbreviations: control + A, control capsules plus vitamin A (vitamin A given as retinyl palmitate); DHA + A, docosahexaenoic acid plus vitamin A; ERG, electroretinogram; ETDRS, Early Treatment Diabetic Retinopathy Study; HFA, Humphrey Field Analyzer. *Data are given as mean ± SE (number of patients sampled). †Differing sample sizes reflect occasional instances where test results were not available at one or more visits for a given outcome variable. ‡P value given for PROC MIXED (of SAS version 6.12) analysis. §Total field sensitivity indicates 30-2 and 30/60-1 total point scores combined when both are available. ࿣Derived from 100 ϫ [1−exp (mean log change)].

30-Hz ERG amplitude, and of ETDRS visual acuity for field sensitivity to the HFA 30-2 and 30/60-1 programs the DHA+A group vs the control+A group among the combined (size V target), 10% of remaining 30-Hz cone 208 patients followed up for each of 4 annual visits. No ERG amplitude, and 0.7 letter of ETDRS visual acuity per significant differences in rates of change were observed year. No significant toxic adverse effects were noted over between these 2 groups. Both groups lost, on average, the course of this trial both for docosahexaenoic acid or about 37 to 38 dB per year to the HFA 30-2 program con- vitamin A supplementation. Similarly, docosahexae- dition and 57 to 60 dB to the HFA 30-2 and 30/60-1 pro- noic acid supplementation (400 mg/d) was associated with grams combined. These total point score declines cor- no identifiable safety risks over 4 years among 23 males respond to losses of approximately 0.5 dB and 0.4 dB per with X-linked retinitis pigmentosa.55 No significant treat- year, respectively, for an average location in the visual ment effect was noted within any of the genetic types. field. Over 4 years, analysis of 30-Hz ERGs showed that Serum triglyceride levels increased by about 25% in the the mean annual rates of decline of remaining function control+A group as described previously.45 In both groups were 9.92% in the DHA+A group and 10.49% in the the high-density lipoprotein cholesterol level increased control+A group, which was not statistically signifi- and the low-density lipoprotein cholesterol level de- cantly different (P=.64). These analyses were also per- creased or remained stable. On balance, changes in these formed on all available data, including patients with par- values would not increase the risk for heart disease. tial follow-up, but with missing values left as missing and Good separation as monitored by mean RBC PE DHA after using multiple imputation methods to account for levels was achieved between the DHA+A group vs the missing data among patients with incomplete follow- control+A group. The DHA+A group had a mean RBC up. No substantive differences were noted between re- PE DHA level 2- to 3-fold greater than the control+A group sults obtained with these additional approaches com- at 1 year and 4 years. At follow-up, averaging years 1 and pared with those in Table 5. A similar analysis as in Table 4, the mean RBC PE DHA level increased by 0.51% from 5 was performed for 0.5-Hz ERG amplitudes based on baseline for the control+A group (significantly different the 55% of patients with quantifiable responses; mean from zero, PϽ.001) and increased 7.95% for the DHA+A rate of decline for the DHA+A group (11.8%) was not group. How this RBC PE DHA change in the control+A significantly different from that for the control+A group group may have influenced their results and, thus, the com- (10.4%) (P=.54). parison of results to the DHA+A group is unclear. We also performed randomized comparisons by ge- The lack of a significant difference in rate of loss of netic type and by dietary intake of ␻-3 fatty acids. No ocular function between the DHA+A and control+A significant differences by treatment group assignment were groups precludes any general recommendation of DHA observed for either the primary or the secondary out- supplementation for patients with retinitis pigmentosa come measures within the dominant, recessive, X- receiving vitamin A. Further investigation of these data linked, or isolate forms of retinitis pigmentosa or within from this trial or other trials may help to clarify whether the category of baseline dietary ␻-3 fatty acid intake (above any subgroups of patients with retinitis pigmentosa will and below intake of 0.16 g/d) (data not shown). benefit from a combination of docosahexaenoic acid plus vitamin A supplementation. COMMENT Submitted for publication July 11, 2003; final revision re- The present trial shows that in adult patients with reti- ceived January 8, 2004; accepted April 19, 2004. nitis pigmentosa, assigned to 15000 IU/d of vitamin A, From the Berman-Gund Laboratory for the Study of a daily supplement of 1200 mg of docosahexaenoic acid Retinal Degenerations, Harvard Medical School, Massa- over a 4-year interval does not, on average, slow the course chusetts Eye and Ear Infirmary, Boston (Drs Berson, Ros- of retinal degeneration. Both the DHA+A and control+A ner, Sandberg, Brockhurst, and Gaudio and Mss Weigel- groups lost 37 to 38 dB of visual field sensitivity to the DiFranco and Anderson); Kennedy Krieger Institute, HFA 30-2 program (size V target), 58 to 60 dB of visual Peroxisomal Diseases Laboratory, Baltimore, Md (Ms

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1303

©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Moser); Foster Biomedical Research Laboratory, Brandeis 17. Berson EL, Rosner B, Sandberg MA, et al. A randomized trial of vitamin A and University, Waltham, Mass (Dr Hayes); Devers Eye Insti- vitamin E supplementation for retinitis pigmentosa. Arch Ophthalmol. 1993;111: 761-772. tute, Portland, Ore (Dr Johnson); the Department of Nutri- 18. Converse CA, Hammer HM, Packard CJ, Shepherd J. Plasma lipid abnormalities tion, Harvard School of Public Health, Boston (Dr Wil- in retinitis pigmentosa and related conditions. Trans Ophthalmol Soc U K. 1983; lett); and the Lipid Metabolism Laboratory, Jean Mayer US 103:508-512. Department of Agriculture Human Nutrition Research, Cen- 19. Bazan NG, Scott BL, Reddy TS, Pelias MZ. Decreased content of docosahexae- ter on Aging at Tufts University, Boston (Dr Schaefer). The noate and arachidonate in plasma phospholipids in Usher’s syndrome. Biochem Biophys Res Comm. 1986;141:600-604. authors have no relevant financial interest in this article. 20. Anderson RE, Maude MB, Lewis RA, Newsome DA, Fishman GA. Fatty acid lev- This study was supported by grant U10EY11030 from els in retinitis pigmentosa [letter]. Exp Eye Res. 1987;44:155-159. the National Eye Institute, Bethesda, Md, and in part by the 21. Gong J, Rosner B, Rees DG, Berson EL, Weigel-Difranco CA, Schaefer EJ. Plasma Foundation Fighting Blindness, Owings Mills, Md. docosahexaenoic acid levels in various genetic forms of retinitis pigmentosa. In- We thank the study patients and their families and grate- vest Ophthalmol Vis Sci. 1992;33:2596-2602. 22. McColl AJ, Converse CA. Lipid studies in retinitis pigmentosa. Prog Lipid Res. fully acknowledge the following individuals who contrib- 1995;34:1-16. uted to the conduct of this trial: Tina Skop-Chaput, Michele 23. Hoffman DR, Birch DG. Docosahexaenoic acid in red blood cells of patients with Berry, Melissa Stillberger, Peggy Rodriguez, Kevin McDer- X-linked retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1995;36:1009-1018. mott, Linda Berard, Heather Lee, Susana Chung, Shyana 24. Schaefer EJ, Robins SJ, Patton GM, et al. Red blood cell membrane phosphati- Harper, Anna Maria Baglieri, Ciara Rice, Cathy Loner- dylethanolamine fatty acid content in various forms of retinitis pigmentosa. J Lipid Res. 1995;36:1427-1433. gan, Suzanne Dalton, Marion McPhee, Martin Van Den- 25. Anderson RE, Andrews LD. Biochemistry of retinal photoreceptor membranes burgh, Anita Liu, David Jones, and Sherrie Kaplan, PhD. in vertebrates and invertebrates. In: Westfall J, ed. Visual Cells in Evolution. New Members of the Data and Safety Monitoring Commit- York, NY: Raven Press; 1975:1-22. tee were Marian Fisher, PhD (chair); George Bresnick, MD; 26. Fliesler SJ, Anderson RE. Chemistry and metabolism of lipids in the vertebrate Baruch A. Brody, PhD; Barry Davis, MD, PhD; Natalie retina. Prog Lipid Res. 1983;22:79-131. 27. Anderson RE, Benolken RM, Dudley PA, Landis DJ, Wheeler TG. Polyunsatu- Kurinij, PhD (ex officio); Carol M. Mangione, MD; James rated fatty acids of the photoreceptor membrane. Exp Eye Res. 1982;18:205- Olson, PhD (1996-1999); Peter R. Pavan, MD (2000- 213. 2003); Sander J. Robins, MD; Pamela Sample, PhD; and Bar- 28. Sarkadi-Nagy E, Wijendran V, Diau G, et al. The influence of prematurity and long bara A. Underwood, PhD (2001-2003). chain polyunsaturate supplementation in 4-week adjusted age baboon neonate Correspondence: Eliot L. Berson, MD, Berman-Gund brain and related tissues. Pediatr Res. 2003;54:244-252. 29. Roelofson B, Op den Kamp JA. Introduction to membrane phospholipids: struc- Laboratory for the Study of Retinal Degenerations, 243 ture, organization, and function. In: Zwaal RFA, ed. Coagulation and Lipids. Boca Charles St, Boston, MA 02114. Raton, Fla; CRC Press; 1988:14-40. 30. Benga G, Holmes RP. Interactions between components in biological membranes and their implications for membrane function. Prog Biophys Mol Biol. REFERENCES 1984;43:195-257. 31. Bazan NG, Burton SL. Docosahexaenoic acid and inherited retinal degenera- 1. Leber T. Die Pigmentdegeneration der Netzhaut und mit ihr verwandte tions. In: Hollyfield JG, Anderson RE, LaVail MM, eds. Progress in Clinical Bio- Erkra¨nkungen In: Graefe A, Saemisch-Hess T, eds. Handbuch der gesammten logical Research. New York, NY: Alan R Liss Inc; 1987:103-118. Augenheilkunde. Vol 7. 2nd ed. Leipzig, Germany: W Engelmann; 1916:1076- 32. Treen M, Uauy R, Jameson DM, Thomas VL, Hoffman DR. Effect of docosa- 1225. hexaenoic acid on membrane fluidity and function in intact cultured Y-79 reti- 2. Bell J. Retinitis pigmentosa and allied diseases of the eye. In: Pearson K, ed. Trea- noblastoma cells. Arch Biochem Biophys. 1992;294:564-570. sury of Human Inheritance. Vol 2. London, England: Cambridge University Press; 33. Mitchell DC, Straume M, Litman BJ. Role of sn-1-saturated, sn-2-poly- 1922:1-28. unsaturated phospholipids in control of membrane receptor conformational equi- 3. Ammann F, Klein D, Franceschetti A. Genetic and epidemiological investigation librium: effects of cholesterol and acyl chain unsaturation on the metarhodop- of pigmentary degeneration of the retina and allied disorders in Switzerland. sin in equilibrium with metarhodopsin II equilibrium. Biochemistry. 1992;31: J Neurol Sci. 1965;2:183-196. 662-670. 4. Boughman JA, Conneally PM, Nance WE. Population genetic studies of retinitis 34. Chen Y, Noy N. Retinoid specificity of interphotoreceptor retinoid-binding pro- pigmentosa. Am J Hum Genet. 1980;32:223-235. tein. Biochemistry. 1994;33:10658-65. 5. Bundy S, Crews SJ. Wishes of patients with retinitis pigmentosa concerning ge- 35. Chen Y, Houghton LA, Brenna JT, Noy N. Docosahexaenoic acid modulates the netic counseling. J Med Genet. 1982;19:317-318. interactions of the interphotoreceptor retinoid-binding protein with 11-cis- 6. Hu DN. Genetic aspects of retinitis pigmentosa in China. Am J Med Genet. 1982; retinal. J Biol Chem. 1996;271:20507-20515. 12:51-56. 36. Chen Y, Saari JC, Noy N. Interactions of all-trans-retinol and long-chain fatty ac- 7. Bunker CH, Berson EL, Bromley WC, Hayes RB, Roderick TH. Prevalence of reti- ids with interphotoreceptor retinoid-binding protein. Biochemistry. 1993;32: nitis pigmentosa in Maine. Am J Ophthalmol. 1984;97:357-365. 11311-11318. 8. Heckenlively JR. Retinitis Pigmentosa. Philadelphia, Pa: JB Lippincott Co; 37. Wolf G. Transport of retinoids by the interphotoreceptor retinoid-binding pro- 1988. tein. Nutr Rev. 1998;56:156-158. 9. Haim M, Holm NV, Rosenberg T. Prevalence of retinitis pigmentosa and allied 38. Endres S, Ghorbani R, Kelley VE, et al. The effect of dietary supplementation with disorders in Denmark. Acta Ophthalmol (Copenh). 1992;70:178-186. n–3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor ne- 10. Berson EL. Retinitis pigmentosa: the Friedenwald Lecture. Invest Ophthalmol Vis crosis factor by mononuclear cells. N Engl J Med. 1989;320:265-271. Sci. 1993;34:1659-1676. 39. Neuringer M, Connor WE, Van Petten C, Barstad L. Dietary omega-3 fatty acid 11. Mandelbaum J. Dark adaptation: some physiologic and clinical considerations. deficiency and visual loss in infant rhesus monkeys. J Clin Invest. 1984;73:272- Arch Ophthalmol. 1941;26:203-239. 276. 12. Sloan LL. Light sense in pigmentary degeneration of the retina. Arch Ophthal- 40. Neuringer M, Anderson GJ, Connor WE. The essentiality of ␻-3 fatty acids for mol. 1942;28:613-631. the development and function of the retina and brain. Annu Rev Nutr. 1988;8: 13. Karpe G. Basis of clinical electroretinography. Acta Ophthalmol (Copenh). 1945; 517-541. 24(suppl):84. 41. Birch DG, Birch EE, Hoffman DR, Uauy RD. Retinal development in very low- 14. Bjork A, Karpe G. The electroretinogram in retinitis pigmentosa. Acta Ophthal- birth-weight infants fed diets differing in omega-3 fatty acids. Invest Ophthal- mol (Copenh). 1951;29:361-376. mol Vis Sci. 1992;33:2365-2376. 15. Verhoeff FH. Microscopic observations in a case of retinitis pigmentosa. Arch 42. Willett WC, Sampson L, Browne ML, et al. The use of a self-administered ques- Ophthalmol. 1931;5:392-407. tionnaire to assess diet four years in the past. Am J Epidemiol. 1988;127:188- 16. Cogan DG. Symposium: primary chorioretinal aberrations with night blindness: 199. pathology. Trans Am Acad Ophthalmol Otolaryngol. 1950;54:629-661. 43. London SJ, Sacks FM, Caesar J, Stampfer MJ, Siguel E, Willett WC. Fatty acid

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1304

©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 composition of subcutaneous adipose tissue and diet in postmenopausal US 49. Sandberg MA, Weigel-DiFranco C, Rosner B, Berson EL. The relationship be- women. Am J Clin Nutr. 1991;54:340-345. tween visual field size and electroretinogram amplitude in retinitis pigmentosa. 44. Moser AB, Jones DS, Raymond GV, Moser HW. Plasma and red blood cell fatty Invest Ophthalmol Vis Sci. 1996;37:1693-1698. acids in peroxisomal disorders. Neurochem Res. 1999;24:187-197. 50. Ferris FL, Kassoff A, Bresnick GH, Bailey I. New visual acuity charts for clinical 45. Sibulesky L, Hayes KC, Pronczuk A, Weigel-DiFranco C, Rosner B, Berson EL. research. Am J Ophthalmol. 1982;94:91-96. Safety of less than 7,500 RE/day (25,000 IU/day) of vitamin A in adults with reti- 51. O’Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biomet- nitis pigmentosa. Am J Clin Nutr. 1999;69:656-663. rics. 1979;35:549-556. 46. Mills RP, Barnebey HS, Migliazzo CV, Li Y. Does saving time using FASTPAC or 52. SAS Institute Inc. SAS Version 6.12. Cary, NC: SAS Institute; 1996. suprathreshold testing reduce quality of visual fields? Ophthalmology. 1994; 53. Diggle P, Heagerty P, Liang K-Y, Zeger S, eds. Analysis of Longitudinal Data. 101:1596-1603. Oxford, England: Oxford University Press; 2002. 47. Schaumberger M, Schafer B, Lachenmayr BJ. Glaucomatous visual fields: FASTPAC 54. Little RJ, Rubin DB. Statistical Analysis With Missing Data. New York, NY: John versus full threshold strategy of the Hymphrey field analyzer. Invest Ophthalmol Wiley & Sons; 1987. Vis Sci. 1995;36:1390-1397. 55. Wheaton DH, Hoffman DR, Locke KG, Watkins RB, Birch DG. Biological safety 48. Flanagan JG, Moss ID, Wild JM, et al. Evaluation of FASTPAC: a new strategy for assessment of docosahexaenoic acid supplementation in a randomized clini- threshold estimation with the Humphrey field analyzer. Graefes Arch Clin Exp cal trial for X-linked retinitis pigmentosa. Arch Ophthalmol. 2003;121:1269- Ophthalmol. 1993;231:465-469. 1278.

Other Species

Leopard Cat. A vertical section of the Bouin-fixed globe of a leopard cat, a nocturnal feline. The very large cornea and the posteriorly positioned, very round lens are features of eyes adapted for nocturnal vision. Courtesy of Richard R. Dubielzig, DVM.

(REPRINTED) ARCH OPHTHALMOL / VOL 122, SEP 2004 WWW.ARCHOPHTHALMOL.COM 1305