Docosahexaenoic Acid Slows Visual Field Progression in X-Linked Retinitis Pigmentosa: Ancillary Outcomes of the DHAX Trial.

PURPOSE: Docosahexaenoic acid (DHA) was supplemented in a single-site, placebo-controlled, randomized clinical trial designed to slow vision loss associated with X-linked retinitis pigmentosa (XLRP); the DHAX Trial. We previously reported no significant differences between supplemented and placebo groups in intent-to-treat analysis of primary ERG outcomes. Assessed herein are hypothesis-generating measures of ancillary visual function outcomes in participants fully adhering to trial protocol. METHODS: Male participants with XLRP (range, 7-31 years) received 30 mg DHA/kg/d (n = 29) or placebo (n = 22) for 4 years. Visual outcomes were measured annually and red blood cell (RBC) DHA determined every 6 months. RESULTS: Oral DHA supplementation increased mean RBC-DHA levels by 4-fold (P < 0.0001) over placebo. No group differences in progression were found for visual acuity (P = 0.11), shape discrimination (P = 0.18), or fundus appearance (P = 0.70). Optical coherence tomography (OCT) became available during year 2 of the trial; no group differences were seen in ellipsoid zone constriction (P = 0.87) over 2 years. Yearly rates of progression were reduced for dark-adapted thresholds (P = 0.06) and visual field sensitivity for foveal, macular, peripheral, total, and ellipsoid zone regions by DHA supplementation (P = 0.039, P = 0.031, P < 0.0001, P < 0.0001, and P = 0.033). Rates of visual field sensitivity decline were dependent on RBC-DHA (P = 0.046 to <0.0001). CONCLUSIONS: Supplementation of DHA significantly elevated blood DHA levels and reduced the rate of progression in final dark-adapted thresholds and visual field sensitivity. From the relationship between RBC-DHA and the rate of field sensitivity loss, we can extrapolate that an RBC-DHA level of 17% could minimize the decline in field sensitivity. (ClinicalTrials.gov number, NCT00100230.)

Safety assessment of docosahexaenoic acid in X-linked retinitis pigmentosa: the 4-year DHAX trial.

PURPOSE: Docosahexaenoic acid (DHA) continues to be evaluated and recommended as treatment and prophylaxis for various diseases. We recently assessed efficacy of high-dose DHA supplementation to slow vision loss in patients with X-linked retinitis pigmentosa (XLRP) in a randomized clinical trial. Because DHA is a highly unsaturated fatty acid, it could serve as a target for free-radical induced oxidation, resulting in increased oxidative stress. Biosafety was monitored during the 4-year trial to determine whether DHA supplementation was associated with identifiable risks. METHODS: Males (n = 78; 7-31 years) meeting entry criteria were enrolled. The modified intent-to-treat cohort (DHA = 33; placebo = 27) adhered to the protocol ≥ 1 year. Participants were randomized to an oral dose of 30 mg/kg/d DHA or placebo plus a daily multivitamin. Comprehensive metabolic analyses were assessed for group differences. Treatment-emergent adverse events including blood chemistry metabolites were recorded. RESULTS: By year 4, supplementation elevated plasma and red blood cell-DHA 4.4- and 3.6-fold, respectively, compared with the placebo group (P < 0.00001). Over the trial duration, no significant differences between DHA and placebo groups were found for vitamin A, vitamin E, platelet aggregation, antioxidant activity, lipoprotein cholesterol, or oxidized LDL levels (all P > 0.14). Adverse events were transient and not considered severe (e.g., gastrointestinal [GI] irritability, blood chemistry alterations). One participant was unable to tolerate persistent GI discomfort. CONCLUSIONS: Long-term, high-dose DHA supplementation to patients with XLRP was associated with limited safety risks in this 4-year trial. Nevertheless, GI symptoms should be monitored in all patients taking high dose DHA especially those with personal or family history of GI disturbances. (ClinicalTrials.gov number, NCT00100230.).

Four-year placebo-controlled trial of docosahexaenoic acid in X-linked retinitis pigmentosa (DHAX trial): a randomized clinical trial.

IMPORTANCE: X-linked retinitis pigmentosa is a severe inherited retinal degenerative disease with a frequency of 1 in 100,000 persons. Because no cure is available for this orphan disease and treatment options are limited, slowing of disease progression would be a meaningful outcome. OBJECTIVE: To determine whether high-dose docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid, slows progression of X-linked retinitis pigmentosa measured by cone electroretinography (ERG). DESIGN, SETTING, AND PARTICIPANTS: A 4-year, single-site, randomized, placebo-controlled, double-masked phase 2 clinical trial at a research center specializing in medical retina. Seventy-eight male patients diagnosed as having X-linked retinitis pigmentosa were randomized to DHA or placebo. Data were omitted for 2 patients with non-X-linked retinitis pigmentosa and 16 patients who were unable to follow protocol during the first year. The remaining participants were tested annually and composed a modified intent-to-treat cohort (DHA group, n = 33; placebo group, n = 27). INTERVENTIONS: All participants received a multivitamin and were randomly assigned to oral DHA (30 mg/kg/d) or placebo. MAIN OUTCOMES AND MEASURES: The primary outcome was the rate of loss of cone ERG function. Secondary outcomes were rod and maximal ERG amplitudes and cone ERG implicit times. Capsule counts and red blood cell DHA levels were assessed to monitor adherence. RESULTS: Average (6-month to 4-year) red blood cell DHA levels were 4-fold higher in the DHA group than in the placebo group (P < .001). There was no difference between the DHA and placebo groups in the rate of cone ERG functional loss (0.028 vs 0.022 log µV/y, respectively; P = .30). No group differences were evident for change in rod ERG (P = .27), maximal ERG (P = .65), or cone implicit time (no change over 4 years). The rate of cone loss (ie, event rate) was markedly reduced compared with rates in previous studies. No severe treatment-emergent adverse events were found. CONCLUSIONS AND RELEVANCE: Long-term DHA supplementation was not effective in slowing the loss of cone or rod ERG function associated with X-linked retinitis pigmentosa. Participant dropout and lower-than-expected disease event rate limited power to detect statistical significance. A larger sample size, longer trial, and attainment of a target blood DHA level (13%) would be desirable. While DHA supplementation at 30 mg/kg/d does not present serious adverse effects, routine monitoring of gastrointestinal tolerance is prudent. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00100230.

Fluorescence biosensing in nanopores.

Hydrated nanopores offer a unique environment for studying biological molecules under controlled conditions and fabricating sensors using fluorescence. Silica nanopores for example are non-toxic, biologically and optically compatible with protein, and can be easily synthesized to entrap protein and exclude potentially interfering macromolecules, while transmitting analytes of interest. A well known problem when polymerizing orthosilicates to fabricate silica sol-gel nanopores is the release of alcohol, which denatures proteins. We will describe how using the fluorescence of PRODAN (6-propionyl-2-(N,N-dimethylamino) naphthalene) to monitor methanol generated during polymerization has helped define a protocol with enhanced biocompatibility. The improved biocompatibility of sol-gel nanopores synthesized using tetramethyl orthosilicate (TMOS) has been demonstrated by preserving the unstable native trimer form of allophycocyanin (APC) for up to 500 Hrs without the need to covalently binding the subunits together. This has enabled the observation of native APC trimer by means of its fluorescence in a pore down to the single molecule level. In this paper we demonstrate how PRODAN and another polarity sensitive dye, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, Nile red (NR) report on pore polarity and successfully extend protein encapsulation to nano-channels of alumina (Al2O3). Improved biocompatibility of nanopores has potential impact in nanomedicine where the ability to study single biomolecules is a primary goal as it underpins our understanding of disease pathology and therapeutics at the most fundamental level. In sensing also the advantages of nanopore isolation of metabolite-specific protein for detecting non-fluorescent metabolites has been demonstrated. Similar approaches can in principle be developed for both single-molecules and lab-on-a-chip sensors.

The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Visual maturation of term infants fed long-chain polyunsaturated fatty acid-supplemented or control formula for 12 mo.

BACKGROUND: Several studies found a benefit of long-chain polyunsaturated fatty acid (LCP) supplementation for visual or mental development, but others found no benefit. Likely contributors to differences among studies are the amount of LCP supplementation, functional outcomes, and sample size. OBJECTIVE: We evaluated LCP supplementation in amounts typical for human milk (based on local and worldwide surveys) in a large cohort of infants by using sweep visual evoked potential (VEP) acuity as the functional outcome. DESIGN: The study was a double-masked, randomized, controlled clinical trial in 103 term infants. By age 5 d, infants were randomly assigned to receive either formula with no docosahexaenoic acid (DHA) or arachidonic acid (ARA) or formula supplemented with DHA and ARA as 0.36% and 0.72%, respectively, of total fatty acids. Sweep VEP acuity was the primary outcome. Random dot stereoacuity, blood lipid profile, growth, and tolerance were secondary outcomes. RESULTS: VEP acuity in the LCP-supplemented group was significantly better than that in the control group at ages 6, 17, 26, and 52 wk. Stereoacuity in the LCP-supplemented group was significantly better than that in the control group at age 17 wk but not at ages 39 and 52 wk. By ages 17 and 39 wk, the red blood cell DHA concentration in the LCP-supplemented group was more than double and more than triple, respectively, that in the control group. Growth of infants fed LCP-supplemented and control formulas did not differ significantly, and both diets were well tolerated. CONCLUSION: LCP supplementation of term infant formula during the first year of life yields clear differences in visual function and in total red blood cell lipid composition.

A randomized, placebo-controlled clinical trial of docosahexaenoic acid supplementation for X-linked retinitis pigmentosa.

PURPOSE: Low docosahexaenoic acid (DHA) in X-linked retinitis pigmentosa (XLRP) may influence retinal function. The goals of this study were to elevate blood DHA levels and determine the effect on the rate of disease progression. DESIGN: In a 4-year prospective randomized clinical trial, male patients with XLRP (mean age = 16 years; range = 4-38 years) received DHA (400 mg/d; n = 23; +DHA group) or placebo (n = 21) capsules. METHODS: Red blood cell (RBC)-DHA concentrations were assessed every 6 months. Full-field cone electroretinograms (ERGs; the primary outcome measure), visual acuity, dark-adaptation, visual fields, rod ERGs, and fundus photos were recorded annually. RESULTS: In the +DHA group, RBC-DHA increased 2.5-fold over placebo levels (70 vs 28 mg DHA/l). Repeated measures analysis of variance for cone ERG showed a significant main effect of year (P <.0001) but not of group (P =.16). Preservation of cone ERG function correlated with RBC-DHA (P =.018), and there was less change in fundus appearance in the +DHA group (P =.04). Neither visual acuity nor visual fields were changed. In subset analysis, DHA supplementation was beneficial in reducing rod ERG functional loss in patients aged <12 years (P =.040) and preserving cone ERG function in patients > or =12 years (P =.038). CONCLUSIONS: Although DHA-supplemented patients had significantly elevated mean RBC-DHA levels, the rate of cone ERG functional loss was not significantly different between groups. Supplemental analyses provided evidence for a DHA benefit and a direction for subsequent investigations.

Aqueous solutions of uranium(VI) as studied by time-resolved emission spectroscopy: a round-robin test.

Results of an inter-laboratory round-robin study of the application of time-resolved emission spectroscopy (TRES) to the speciation of uranium(VI) in aqueous media are presented. The round-robin study involved 13 independent laboratories, using various instrumentation and data analysis methods. Samples were prepared based on appropriate speciation diagrams and, in general, were found to be chemically stable for at least six months. Four different types of aqueous uranyl solutions were studied: (1) acidic medium where UO2(2+)aq is the single emitting species, (2) uranyl in the presence of fluoride ions, (3) uranyl in the presence of sulfate ions, and (4) uranyl in aqueous solutions at different pH, promoting the formation of hydrolyzed species. Results between the laboratories are compared in terms of the number of decay components, luminescence lifetimes, and spectral band positions. The successes and limitations of TRES in uranyl analysis and speciation in aqueous solutions are discussed.

Biological safety assessment of docosahexaenoic acid supplementation in a randomized clinical trial for X-linked retinitis pigmentosa.

BACKGROUND: In a 4-year placebo-controlled trial to elevate blood docosahexaenoic acid levels in patients with X-linked retinitis pigmentosa (XLRP), the goal was to assess the potential benefit of docosahexaenoic acid supplementation in altering disease progression. However, docosahexaenoic acid (22:6omega3) is a highly unsaturated fatty acid and considered a target molecule for free-radical oxidative damage. Thus, nutritional provision of docosahexaenoic acid might lead to an increase in antioxidant stress. Additional concerns, such as decreased platelet aggregation, increased bleeding time, and alterations in lipoprotein cholesterol levels, have been reported in supplementation studies with long-chain polyunsaturates. OBJECTIVE: To assess the biological safety of long-term docosahexaenoic acid supplementation. DESIGN: Forty-four male patients (mean age, 16 years) enrolled in a randomized, double-masked, clinical trial and received docosahexaenoic acid, 400 mg/d, or placebo. Blood samples were collected every 6 months. Biological safety analysis included fatty acids, vitamin A and E concentrations, antioxidant capacity, platelet aggregation, alanine aminotransferase activity, and lipoprotein cholesterol and triglyceride profiles. RESULTS: Mean plasma docosahexaenoic acid levels were elevated 2.5-fold by supplementation compared with baseline. Patients receiving placebo capsules exhibited no change (P =.35) in plasma docosahexaenoic acid content. All adverse events reported were minor and equivalently distributed between groups. Plasma vitamin A concentrations remained unchanged during the trial. Mean plasma vitamin E concentrations were correlated with age (P =.005), such that as patients with XLRP matured, plasma vitamin E concentrations increased to approach normal values. There was a trend (P =.10) toward lower mean vitamin E concentrations in the docosahexaenoic acid-supplemented group after 4 years. Docosahexaenoic acid supplementation did not compromise plasma antioxidant capacity, platelet aggregation, liver function enzyme activity, or plasma lipoprotein lipid content in patients with XLRP. CONCLUSION: Long-term docosahexaenoic acid supplementation to patients with XLRP was associated with no identifiable safety risks in this 4-year clinical trial.

Visual function in breast-fed term infants weaned to formula with or without long-chain polyunsaturates at 4 to 6 months: a randomized clinical trial.

OBJECTIVE: Breast-fed infants receive docosahexaenoic acid (DHA) and arachidonic acid (ARA) in their diet. Upon weaning, infants lose this dietary source of long-chain polyunsaturates because many commercial formulas do not contain these important constituents for neural membrane biogenesis. We evaluated the benefits of postweaning dietary supplementation of DHA + ARA on visual maturation. STUDY DESIGN: Healthy term infants (n = 61) were breast-fed to 4 to 6 months, then were randomly assigned to commercial formula or formula supplemented with DHA (0.36%) + ARA (0.72%). Measurements of red blood cell (RBC) fatty acids, visually evoked potential (VEP) acuity, and stereoacuity were done before and after weaning. RESULTS: At 1 year of age, RBC-DHA in the commercial formula-fed group was reduced by 50% from the weaning level, whereas there was a 24% increase in the DHA + ARA-supplemented group. The primary outcome measure, VEP acuity, was significantly more mature in supplemented infants at 1 year of age. Elevated RBC-DHA levels were associated with more mature VEP acuity. There were no significant diet-related differences in stereoacuity. CONCLUSIONS: These data extend through the first year of life the critical period in which a dietary supply of DHA and ARA can contribute in optimizing visual development in term infants.

A randomized controlled trial of long-chain polyunsaturated fatty acid supplementation of formula in term infants after weaning at 6 wk of age.

BACKGROUND: The critical period during which the dietary supply of long-chain polyunsaturated fatty acids (LCPs) may influence the maturation of cortical function in term infants is unknown. OBJECTIVE: The aim of the present study was to determine the relative importance for maturation of the visual cortex of the dietary supply of LCPs during the first 6 wk of life compared with that during weeks 7-52. DESIGN: A randomized controlled clinical trial of LCP supplementation in 65 healthy term infants who were weaned from breast-feeding at 6 wk of age was conducted to determine whether the dietary supply of LCPs after weaning influenced the maturation of visual acuity and stereoacuity. RESULTS: Despite a dietary supply of LCPs from breast milk during the first 6 wk of life, infants who were weaned to formula that did not provide LCPs had significantly poorer visual acuity at 17, 26, and 52 wk of age and significantly poorer stereoacuity at 17 wk of age than did infants who were weaned to LCP-supplemented formula. Better acuity and stereoacuity at 17 wk was correlated with higher concentrations of docosahexaenoic acid in plasma. Better acuity at 52 wk was correlated with higher concentrations of docosahexaenoic acid in plasma and red blood cells. No significant effects of diet on growth were found. CONCLUSION: The results suggest that the critical period during which the dietary supply of LCPs can influence the maturation of cortical function extends beyond 6 wk of age.