Author ORCID Identifier
https://orcid.org/0000-0001-8107-4201
Document Type
Dataset
Start Date
2019
Disciplines
Optics
End Date
2023
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
Recommended Citation
Udensi, J., Loskutova, E., Loughman, J., & Byrne, H. J. (2024). Analysis of macular pigment carotenoids in human blood serum of glaucoma patients as a measure of ocular health: A Raman spectroscopic study [Data set]. Technological University Dublin. DOI: 10.21427/RR9X-EP08
DOI
https://doi.org/10.21427/RR9X-EP08
Methodology
- 2. Materials and methods
2.1 The ENIGMA Study
The ENIGMA study [1], (registered at ClinicalTrials.gov, identifier NCT04460365) was a randomised, placebo-controlled, double-masked clinical trial, designed to evaluate the Macular Pigment response of patients with open angle glaucoma (OAG) to supplementation with lutein (10 mg), zeaxanthin (2 mg), and meso-zeaxanthin (10 mg) over an 18-month period. The supplement ratio was the same as that of commercially available macular pigment supplementation, Macushield, provided by Thompson & Capper Ltd, Runcorn, United Kingdom. The dose was also deemed safe, with renal, liver, lipid, hematologic, and inflammatory biomarkers all unaffected by supplementation at these concentrations [2].
The recruitment was carried out at the Mater Misericordiae University Hospital and Mater Private Hospital (Dublin, Ireland). Study visits were arranged at the Centre for Eye Research Ireland, a dedicated academic clinical trial centre at Technological University Dublin. All necessary research ethical approval were obtained from the Mater Misericordiae Institutional Review Board and Technological University Dublin Research Ethics and Integrity Committee [1]. Written consent forms were supplied by all participants. The study also adhered to the tenets of the Declaration of Helsinki.
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 [3]. These eccentricity regions represent the ocular distribution of the MP carotenoids in the fovea of the retina. Meso-zeaxanthin is situated most centrally at 0.25° This is followed by zeaxanthin, situated in the 0.5° region and then lutein at 1.0° [4]. 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 [5].
The main outcome of the trial was a statistically significant increase in MPOD volume (significant time effect: F(3,111) = 89.31, mean square error (MSE) = 1656.9; P < 0.01), which was observed among those randomised to receive the macular carotenoid supplement over the 18-month trial duration. The study also reported an inverse and statistically significant relationship between baseline MPOD volume and percentage change in MPOD volume over the supplementation period. Notably, the study reported no clinically significant structural or functional changes recorded through the supplementation period.
2.2 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 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 [1].
In total, serum samples from 40 participants of the ENIGMA study were made available for the Raman spectroscopic analysis. Data from all clinical parameters analysed were available for 20 baseline participants samples and 20 eighteen-months supplemented participants samples. These were used for both the combined spectroscopic analysis with baseline and supplemented samples as well as the baseline only/18-month only analysis with 20 patients samples in each set. Furthermore, from the set of 20 baseline/supplemented, matching baseline and 18-months supplemented samples were available for 8 participants only, and this set was also used to carry out the Raman spectroscopic analysis.
2.3 Raman spectroscopy and pre-processing
Raman spectral measurements [3] 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) [6]. 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, as previously described [7].
2.4: Percentage change of serum carotenoids over supplementation period
The relationship between baseline Raman serum measurements and percentage change in carotenoid supplementation over the 18 month period of the ENIGMA study was estimated for the 20 baseline serum samples available for the study, based on the changes in intensity of the Raman signatures of the characteristic carotenoid peaks, specifically at 1004 and 1158 cm-1[8], before and after supplementation (100 x (18 months - Baseline)/Baseline). Baseline and 18 months supplemented Raman measurements from all 40 participants were used in the analysis and percentage change was plotted against baseline Raman serum measurement.
2.5: Partial least squares regression analysis (PLSR) and cross validation
Multivariate regression analysis of the Raman responses was carried out following pre-processing, using partial least-squares regression analysis (PLSR) to examine the correlation of the clinical parameters with the Raman measurements, and explore the predictive capacity of the technique. The PLSR algorithm examines variation in spectral data or predictors, (X matrix), as they relate to the associated factors or responses, (Y matrix), according to the linear equation Y = XB + E, where B is the regression coefficient matrix and E is the residual matrix [9,10]. The Y matrix, or “target” variable is usually a quantifiable or systematically varied external factor, in this case measurements from the clinical parameters. It then attempts to maximise the covariance of X (the Raman spectra) and the target, Y, described according to Latent Variables (LV) in a systematic model [9,10]. In summary, it can reduce the number of predictors to a smaller set of uncorrelated components or latent variables which, cumulatively and progressively ((LV1 > LV2 etc.) account for the co-variance. Least squares regression is therefore carried out on the latent variables, rather than on the original data [9,11].
The loading of the LV reveals the spectral features which contribute to that LV, and therefore to the co-variance. The Regression Co-efficient can be considered the weighted sum of all the contributing LVs, and in spectral analysis, for a good correlation, should yield the spectrum of the constituent components which vary systematically as a function of the target variable.
For this study, PLSR was used to examine the degree of correlation between the ocular parameters measured in the ENIGMA study and the Raman spectroscopic measurements, as represented by the coefficient of determination (R²). The regression was carried out, first using the combined baseline and supplemented serum from all 20 participants, and then using either baseline or supplemented serum and in a set of 20 participants’ samples and in a restricted group of 8 matching participants samples. This was done in order to establish the best correlation from the clinical parameters from the samples made available to the study and is of any particular clinical relevance. The number of LVs used in the regression analysis was chosen by identifying the point at which the cumulative %Variance Explained reached ~100%.
A Leave-One-Patient Out Cross-Validation (LOPOCV) process [12], was employed, such that. all replicate measurements of a patient were grouped and simultaneously removed from the training, to ensure that measurements of the same patient are not used to both train and test the PLSR model. Ultimately the PLSR model constructed can be employed as a predictive model, which can be used, for example, to predict the value of the target variable, based on the spectrum of an unknown sample, or vice versa, with an accuracy described by the Root Mean Squared Error of Prediction (RMSEP) [12,13].
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2. Connolly, E.E.; Beatty, S.; Loughman, J.; Howard, A.N.; Louw, M.S.; Nolan, J.M. Supplementation with All Three Macular Carotenoids: Response, Stability, and Safety. Invest Ophthalmol Vis Sci 2011, 52, 9207–9217, doi:10.1167/IOVS.11-8025.
3. Udensi, J.; Loskutova, E.; Loughman, J.; Byrne, H.J. Quantitative Raman Analysis of Human Blood Serum of Glaucoma Patients Supplemented with Macular Pigment Carotenoids. Datasets 2024, doi:https://doi.org/10.21427/JHC3-G466.
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english
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Technological University Dublin
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