Analysis of macular pigment carotenoids in human blood serum of glaucoma patients as a measure of ocular health: A Raman spectroscopic study

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Abstract Carotenoids are a major component of the human diet and have been widely studied for their antioxidant, and vision protection roles in the human body, and dietary supplementation is promoted in particular for ocular health. An initial trial (European Nutrition in Glaucoma Management (ENIGMA)), which assessed macular pigment optical density (MPOD) as well as ocular structural, functional and perceptual parameters before and after the 18-month supplementation of glaucoma patients with macular pigment (MP) carotenoids (lutein, zeaxanthin, meso-zeaxanthin), confirmed supplementation significantly improved the clinical ocular health of participants. Blood contains all major dietary carotenoids, presenting it as a suitable and efficient alternative medium for analysing dietary carotenoids in vivo. Raman spectroscopy, a proven efficient analytical tool was used to explore the impact of the supplementation on participants’ serum carotenoid levels and any correlations with MPOD and other ocular responses. Samples obtained from the pre-supplementation baseline and 18-month supplemented participants were analysed. An inverse relationship was observed between percentage change in Raman intensity over the supplementation period and the baseline Raman serum measurements, indicating greater relative benefit of carotenoid supplementation for people with low MPOD/serum carotenoids pre-supplementation. Partial least squares regression (PLSR) was employed to analyse the spectra after pre-processing and the loadings reflected the carotenoid content and structural profile. MPOD results correlated at all eccentricities, with a coefficient of determination (R2) of 0.62 – 0.92 and % Root mean squared error <44%. Structural, functional and perceptual parameters analysed also showed good degree of correlation with the serum Raman measurements. The results support the conclusions of the ENIGMA trial and suggests strategies for optimising patient responses to supplementation, based on baseline carotenoid levels, and furthermore points to the significant potential of Raman spectroscopy of serum carotenoids as a simple and reliable alternative method for investigating macular pigment carotenoids and assessment of patient health.

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Ocular measurements (from the ENIGMA trial (registered at ClinicalTrials.gov, identifier NCT04460365) https://doi.org/10.1016/j.xops.2021.100039 62 patients voluntarily participated in the study, of which 42 randomly received the carotenoid supplements, while 20 received the placebo, which contained only sunflower oil. Ocular parameters recorded include Macular pigment optical density volume within the central 6° of retinal eccentricity as well as at 0.23°, 0.51°, 0.74°, and 1.02°, recorded using autofluorescence.Furthermore, visual functional parameters, microperimetry average threshold and visual acuity (VA) were measured alongside structural parameters, macular retina nerve fibre layer thickness (mRNFL), Ganglion cell complex (GCC) and Ganglion cell layer thickness (GCL), which were all measured to assess visual function and glaucoma severity. Lastly, the Glaucoma Activities Limitation 9 questionnaire (GAL 9), which assessed the quality of life of patients, was also analysed as a perceptual parameter. Blood samples Blood samples were collected within the Centre for Eye Research Ireland (CERI) from all 62 participants. Samples were drawn and processed to obtain serum by personnel from CERI, after which they were stored at -80℃ . When needed, then samples were thawed in a water bath at 37℃, and Raman spectroscopic measurements were carried out immediately. Samples were obtained at baseline (before supplementation) and at 18 months (after supplementation), were examined only from participants who had received the carotenoid supplements. Samples from participants who received the placebo were not examined, as the original study reported no observable effects. Raman spectroscopy and pre-processing Raman spectral measurements of the blood samples were carried out using a Horiba Jobin-Yvon LabRam HR800 spectrometer with a 16-bit Peltier cooled CCD detector, coupled to Olympus 1X71 inverted microscope. A 532 nm laser line of ~12 mW at the sample was used in taking measurements with a 300 lines/mm grating, throughout the study. Serum measurements were performed by focussing the laser into the samples contained in a cover slip glass bottom 96-well plate (Matek), using a x60 water immersion objective (LUMPlanF1, Olympus) (49). The spectral range employed was 400 – 4000 cm-1 and the back scattered Raman signal was typically accumulated for 5 x 4 seconds. 4-5 spectra were acquired from different spots on each sample. Pre-processing techniques were then applied to smooth the raw spectra and remove inherent background water and glass contributions before further analysis.

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