Bilirubin is a yellow pigment produced in the body, primarily as a consequent-by-product of red blood cell (RBC) catabolism (Adamson and Longo, 2012). Senile RBCs at the end of their 120 days’ sojourn in circulation, are physiologically detected and hemolysed by the reticulo-endothelial system. This process occurs chiefly in the spleen, causing the release of hemoglobin (Hb), the principle RBC component (Korolnek and Hamza, 2015). When the tetra-pyrrolic moiety of hemoglobin, called heme, breaks down, it generates plasma-insoluble unconjugated bilirubin (UCB) (Schiff et al., 2011). To render it plasma-soluble, UCB is reversibly-bound with plasma albumin and is transported to the liver to undergo a process called conjugation. In the liver, UCB is conjugated with glucuronic acid (Levitt and Levitt, 2014). Plasma-soluble conjugated bilirubin (CB) is then released through the bile into the intestine, from where it is excreted and eliminated from the body through the bowels and bladder in the form of stercobilinogen (Erlinger et al., 2014).

In any circumstances of increased hemolysis, hepatic dysfunction or even biliary obstruction, there ensues a build-up of bilirubin in blood, resulting in a condition termed hyperbilirubinemia, which is clinical jaundice (Fargo et al., 2017). Normal serum bilirubin levels are not known to pose any implications for general health, rather, is found to be valuable for possessing antioxidant properties in the vasculature (Park, 2016). However, uncontrolled and/or untreated hyperbilirubinemia is toxic and can cause serious deleterious effects that require immediate and appropriate life-saving treatments (Hansen, 2001). Currently, there exist numerous reports that describe the lethal effects of jaundice, but most studies are directed toward its effects on the nervous system (Shapiro, 2003; Shi et al., 2019). There exist fewer reports of bilirubin toxicity on blood components. It has been recognized that bilirubin liberated from RBC destruction, if present in larger than physiological quantities in circulation, can trigger toxic effects on other circulating RBCs. This can result in augmenting the production of more bilirubin, the whole course transitioning into a potentially vicious cycle (Lang et al., 2015). In addition to bilirubin, levels of a multitude of inflammatory markers and acute phase reactants escalate in the serum, such as endotoxins, tumor necrosis factor-α, IL-6, and C-reactive protein (Padillo et al., 2001). These systemic inflammatory cytokines are themselves known to reduce the life-span of RBCs, through a programmed process called eryptosis (Pretorius, 2018).

The focus of this study was to investigate the chemometrics of the Raman spectra of RBCs from blood samples of patients with jaundice, keeping the cells as alive as possible. This required the utility of a tool that demands only the bare minimal sample-preparation procedures, reagents or fixatives, which could themselves be potential sources of cell-injury or cell-modification (Sato et al., 2018). Keeping these viewpoints into consideration, we made use of a home-built micro-Raman spectroscopy system coupled with laser-tweezers to investigate the biochemical characteristics of RBCs (Bankapur et al., 2010). Raman spectroscopy has been gaining popularity in its application in biomedicine as this single-cell technique is tremendously sensitive to very subtle molecular changes within the cell, records vibrations of molecular bonds in its original state instantaneously and does not employ the usage of any chemical markers (Kumamoto et al., 2018). This research aimed to comprehend the molecular changes of RBCs, in a “jaundice” environment.

Jaundice (derived from the French word “jaune” which means yellow) is the yellowish discoloration of the sclera, skin and mucous membrane, caused by the build-up of serum bilirubin, above 2 mg/dL, and nearly always signifies the existence of an underlying infection related to the derangement of bilirubin metabolism. Its persistence is associated with a substantial increase in morbidity and mortality (Anand and Garg, 2015). Our body produces close to 4 mg/kg bilirubin every day (Schiff et al., 2011). Normally, the total serum bilirubin level never exceeds 1 mg/dL, of which UCB is less than 0.8 mg/dL and CB levels is less than 0.3 mg/dL (Zieve et al., 1951; Werner et al., 1970). There exists a myriad of clinical pathologies that results in jaundice. Unconjugated hyperbilirubinemia results primarily due to excessive hemolysis, while hyperbilirubinemia caused by excessive CB, develops in hepatocellular diseases and obstructions of the biliary system. In the latter cases, there can be a rise in both forms of bilirubin (Fargo et al., 2017). In this study, we have included patients diagnosed with hyperbilirubinemia, irrespective of its cause.

Hyperbilirubinemia can be toxic to circulating RBCs, causing morphological and metabolic impairments, eventually leading to further hemolysis, aggravating hyperbilirubinemia and intensifying anemia (Lang et al., 2015). Bilirubin toxicity on RBCs has been found to occur in a concentration and temperature-dependent manner (Brito et al., 2000). During the course of the disease, jaundice is accompanied by elevated levels of several enzymes and inflammatory-cytokines in serum (Padillo et al., 2001). This adds up to the already existing distress on healthy RBCs (Pretorius, 2018). Taking these facets into account, we attempted to examine the biochemical status of live RBCs in jaundice employing Raman spectroscopy. Since this analytical technique is both reagent and fixation-free, it rules out any concerns regarding possible cell modifications or damage caused by the analytical processes itself (Sato et al., 2018). Because RBCs are delicate and extremely sensitive cells, it was very important to ensure utmost care in the management of samples during the procedures that might themselves lead to chemometric variations, hemolysis or even eryptosis. We had also ensured to conduct the experiments immediately on sample-procurement.

Our results showed distinct variations in the biochemical signatures of RBCs in jaundice, when compared to the normal. Since RBCs are simple organelle-less cells with a highly deformable cell membrane that holds abundant cytoplasmic Hb, the obtained Raman spectral features were derived from these two sources. The overlaid averaged Raman spectra from the groups displayed Raman peaks that had their intensities altered and a few peaks that had undergone a shift (Figure 2 and Table 1). It is to be noted that the Raman peaks were assigned to specific biochemical components of the RBC in accordance with earlier studies and in this study we report how these Raman signatures altered in clinical jaundice. Spectral analysis and the various multivariate analytical methods employed in this study indicate chemometric modifications of RBCs in jaundice, when compared to RBCs of the healthy group. The variations (section “RBC Raman Markers Detected in Jaundice”) were noted in Raman peaks that represented the “oxygenation status” of Hb. The heme in jaundice exhibited largely a “deoxy” status when compared to the more “oxy” states in the healthy population. This was accompanied by a decline in the frequencies signifying “oxygen saturation” in the jaundice samples. Heme-aggregation-Raman-markers were manifested in jaundice along with a fall in peaks that depicted Hb stability. The spectra also exhibited alterations in Raman peaks portraying RBC membrane stability. The non-parametric MANOVA method served as an aid in the identification of the Raman signatures that could distinctly (p < 0.00001) capture the significant differences in the intensity scores across healthy and jaundice populations (Table 1). PCA of these identified Raman signatures captured better variance between the groups.

These results exhibit enough evidence to establish that in jaundice, intact and healthy-appearing RBCs in circulation, can experience biochemical alterations similar to those seen when under distress. The probable reasons could be the hostile milieu created by the disease itself such as hyperbilirubinemia, presence of pathogens and/or products related to the immune response that alters the physiological state of blood, thereby experiencing a shortened life-span. The intention of this study was to report the Raman spectroscopy features of RBCs in jaundice and analyze the generated findings, irrespective of the disease or it’s cause. An attempt was made to correlate Raman spectral changes with serum bilirubin levels in jaundice. But the results with this group yielded less correlation (Supplementary Figure S2 and Supplementary Table S1). Further exploratory studies are required to investigate the correlation of bilirubin levels and other systemic factors with RBC Raman spectral variations in specific pathologies.

It is challenging to unearth the exact biochemical status of RBCs, as existent in-vivo, considering their extremely delicate and sensitive nature to even minuscule changes in its environment. Therefore, on a routine basis, RBC pathology is clinically assessed by a collective assemblage of tests that encompasses the estimation of its counts, Hb and bilirubin levels and analysis using peripheral smears. Unlike routine assays or even other high-end analytical techniques such as flow cytometry, Raman spectroscopy does not demand labels and is unique by being more sensitive and specific. The Raman-scattered-light from a particular substance characterizes its own exclusive “chemical-fingerprint.” In recent times this tool has been increasingly employed in hematological research for elucidating structural information from hemes, demonstrating the existence of characteristic molecular signatures from them under different conditions of stress such as oxidation and energy depletion, disorders such as thalassemia and diabetes, and diseases such as malaria and carcinomas. This study is merely a commencement to understand the chemometric behavior of RBCs in jaundice and needs to be extrapolated further to comprehend its characteristics in specific diseases/conditions that lead to it.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

The studies involving human participants were reviewed and approved by the Institutional Ethics Committee, Kasturba Medical College and Kasturba Hospital Registration no. ECR/146/Inst/KA/2013/RR-16. The participants provided their written informed consent to participate in this study.

SJ, AB, PR, and SC conceived and designed the study. SJ and AB performed the experiments, interpreted the results, and wrote the manuscript. SB and MA contributed to performing the experiments. RR and PB contributed to the conception and design of the project and the interpretation of results. AK and RL contributed to the statistical analysis, interpretation of results, and wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.