Author ORCID Identifier
Document Type
Dataset
Start Date
2019
Disciplines
Optics, Organic Chemistry, Ophthalmology
End Date
2023
Abstract
Carotenoids have been widely studied for their high dietary, antioxidant, and vision protection roles in the human body. Blood contains all major dietary carotenoids and hence presents as a suitable and efficient substrate for the estimation of dietary carotenoids in vivo. Following the 18-month supplementation of open angle glaucoma patients with macula pigment carotenoids (Lutein, Zeaxanthin and Meso-Zeaxanthin) in the European Nutrition in Glaucoma Management (ENIGMA) trial, Raman spectroscopic analysis of the pre-supplementation baseline and 18-month supplemented blood serum carotenoids from participants was carried out, to investigate the systemic impact of supplementation in the blood and explore a more direct way of quantifying this impact using a routine blood test. A 532 nm laser source was used to provide optimal response. After pre-processing techniques were carried out to remove water and glass contributions, principal components analysis (PCA) was used to differentially analyse serum spectra, of the pre and post-supplementation data sets, a data set of matched pre- and post-supplementation patient samples, and in patients with the highest and lowest carotenoid signals. Following supplementation, a consistent increase in serum carotenoid concentration was observed from the corrected spectra and PC1 loadings, indicating an increase due to the carotenoid supplementation regimen. PCA also revealed differences in the structural profile of the carotenoid content of the two groups, indicated by a shift in the 1519 cm-1 carotenoid peak, consistent with an increased content of the supplemented carotenoids in the blood stream. This increase was also seen to be highest in patients which had a high baseline carotenoid content. The findings highlight the potential of Raman spectroscopy to quantify and differentiate carotenoids directly in blood serum. It could provide guidance for individualised supplementation regime for the management of diseases for which the dietary carotenoids are directly implicated.
Recommended Citation
Udensi, J., Loskutova, E., Loughman, J., & Byrne, H. J. (2024). Quantitative Raman Analysis of Human blood serum of glaucoma patients supplemented with macular pigment carotenoids [Data set]. Technological University Dublin. DOI: 10.21427/JHC3-G466
DOI
https://doi.org/10.21427/JHC3-G466
Methodology
2.1 Patient Samples
Patient serum samples were obtained from the Centre for Eye Research Ireland (CERI). Blood samples were drawn from a total of 63 patients who were voluntary participants in the European Nutrition in Glaucoma Management (ENIGMA) trial, registered at ClinicalTrials.gov, identifier NCT04460365 [58]. Recruitment was carried out at the Mater Misericordiae University Hospital and Mater Private Hospital (Dublin, Ireland), whereas study visits were conducted at the Centre for Eye Research Ireland, a dedicated academic clinical trial centre at Technological University Dublin. All necessary research ethical approval was obtained from the Mater Misericordiae Institutional Review Board and from Technological University Dublin Research Ethics and Integrity Committee [58]. Written consents were obtained from participants, and the study adhered to the tenets of the Declaration of Helsinki.
The ENIGMA study [58] 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. 42 participants randomly received the carotenoid supplements, while 20 received the placebo, which was as a soft gel capsule, identical in appearance to the active supplement, but containing only sunflower oil. Blood samples were taken at the beginning of the study (baseline) and at 6-monthly intervals over 18 months. Macular pigment measurements and other optometric parameters were also recorded.
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). A 60% mean increase in MPOD volume was observed over the 18-month trial duration among those randomised to receive the macular carotenoid supplement, whereas MPOD remained relatively unchanged among those assigned to placebo.
For the present study, blood serum samples obtained at baseline (before supplementation) and at 18 months, from patients who had received the carotenoid supplements and not the placebo, were used for Raman spectroscopic analysis. Blood samples were drawn and processed to obtain serum by personnel from CERI. Samples were stored at -80℃ and thawed in a water bath at 37℃, after which Raman spectroscopic measurements were immediately carried out.
Serum samples from 40 patients were used for the entire Raman analysis. Baseline samples were available for 25 patients and 18 months supplemented samples available for 28 patients. Both baseline and 18-months supplemented samples were available for 8 patients, and this set was used for the matched patient analysis (section 5.3.5c).
1.2 Reference sample preparation
Beta carotene and bovine serum albumin (BSA) powders were purchased from Sigma Aldrich (Arklow, Ireland). The reference samples were prepared by adding 20ml of ultra-pure water (Millipore) to 1g of the powder and mixed until a thick paste was formed. The pastes were measured immediately on a glass slide.
2.3 Raman spectroscopic analysis
Raman spectral measurements 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 and a x60 water immersion objective (LUMPlanF1, Olympus) was employed. A 532 nm laser line of 12 mW was used in taking measurements with a 300 lines/mm grating throughout the study. The spectral range employed was 400 – 4000 cm-1 and the back scattered Raman signal was typically accumulated for 5 x 4 seconds. Depending on the measurement, 4-6 spectra were acquired per sample.
Raman measurements of the beta carotene and BSA used for reference were obtained by measuring a wet paste of the powder of known concentration at room temperature with x60 objective and using a 532 nm laser. Serum measurements were performed by focussing into the samples contained in a cover slip glass bottom 96-well plate (Matek), using the x60 water immersion objective [30].
2.4 Data processing and analysis
All data processing and analysis was conducted using the MATLAB platform. The initial Raman spectroscopic analysis was carried out on the entire data set, including both baseline and 18 month-supplemented blood serum. Blood serum is very complex, typically containing more than 10,000 different proteins [59], as well as other components such as metabolites, peptides, sugars, and lipids [60]. Notwithstanding this, carotenoids (even though found in small quantities in the blood) dominate the spectrum of blood serum, especially at resonant or near resonant conditions [61,62]. A typical Raman spectrum of serum taken with a 532 nm laser source therefore shows a spectral profile of mixed signals from carotenoids, water, serum albumin (as the dominant serum protein), other serum proteins, and stray light scattering [63,64]. The adapted Extended Multiplicative Signal Correction (EMSC) algorithm was used to improve the spectra by performing background subtraction of water [30,65]. It was essential to remove water because it makes up 91 -92% of serum [66], so strongly dominates the spectra of blood serum. This was done by inputting a known spectrum of water into the EMSC algorithm [67]. Before EMSC correction, all spectra were smoothed using the Savitsky-Golay algorithm (polynomial order of 5, window of 9) to reduce the noise. As an extra precaution, the spectrum of glass was also subtracted from the serum spectra. The glass spectrum can contribute to the spectra in the inverted geometry, especially when using very low volume samples [68,69]. Some of the baseline spectra showed interference from the glass peak at 950cm-1 (figure not shown). It was therefore important to remove any possible glass signal from both baseline and the supplemented serum spectra; hence it was subtracted alongside water in the EMSC algorithm by inputting the spectra of the empty glass 96 well plate into the algorithm. The corrected spectra were also water normalised by the co-efficient of the subtracted water, as an internal standard, using the modification of the method by Parachalil et al. [21,70].
Supplemental Figure S1(a) is the 532 nm spectrum of the glass 96 well plate used for measuring the serum samples. Glass based substrates typically show significant Raman scattering contributions in the region of ~500 – 1000 cm-1, originating from SiO2 vibrations [68,69]. Figure S1(b) is the water spectrum, used in the EMSC algorithm to subtract water, which constituted a major part of the background. The spectrum of water shows characteristic peaks at ~1640 cm-1 from the H-O-H scissors, and a broad band from ~3000-3600 cm-1, from the OH symmetric and antisymmetric stretch [52].
Figure S1(c) is the spectra of pure beta carotene (paste) at 532 nm, which was used as reference for the EMSC correction. The spectrum clearly exhibits the characteristic carotenoid peaks at ~1519 cm-1, ~1158 cm-1 and ~1004 cm-1 [40].Also used as reference for the EMSC correction was the spectrum of BSA, in figure S1(d). BSA exhibits typical protein features such as the sharp phenyl alanine peak at ~1004 cm-1, the broad Amide I band at ~ 1620 cm-1, which can be observed in the fingerprint region of 400 – 1800 cm-1 and the broad CH band at 2900 cm-1 [71].
2.5 Principal components analysis
Principal Components Analysis (PCA) is a multivariate analysis technique commonly used to analyse multi-dimensional data sets by reducing the number of variables in data sets with multi dimensions without altering the major variations within the data set [72]. The order of the principal component describes the relevance to the data set. PC1 is expected to describe the highest variation in the data, followed by PC2, PC3 and so on. The first 3 PCs will generally provide up to 99% variance in the data set, giving the best visualisation of the differentiation in the cluster sets. [72,73].
In this study, PCA was used to explore the differences in both carotenoid content and structure of the baseline and 18 months supplemented blood serum spectral data sets. The data used for PCA was already corrected for interferents (water and glass), noise and normalised for water content. PC1 and PC2 loadings were sufficient to show majority of the variation in the data set. The spectral loading of PC1 was used to highlight the difference in the concentration of carotenoids, while PC2 highlighted the difference in carotenoid structures [74].
The spectra are mean centred as an integral part of the PCA algorithm in Matlab, and the effect of vector normalisation in addition to the normalisation to water content on the analysis is also explored.
Language
English
Funder
Technological University Dublin post graduate scholarship 2020
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