Carotenoids: Structures, Functions, and Analysis Methods

What are Carotenoids?

Carotenoids are a group of organic pigments found in plants, algae, bacteria, fungi, and some types of archaea. They are responsible for the vibrant colors observed in fruits, vegetables, flowers, and various other organisms. Carotenoids are characterized by their distinctive chemical structure, which consists of long carbon chains with alternating single and double bonds, often arranged in conjugated systems.

Chemical structures of the carotenoids found most often in the human diet (Ishida et al., 2005).

Structures of Carotenoids

Carotenoids are organic compounds characterized by a series of conjugated double bonds, which give them their distinctive colors ranging from yellow and orange to red. These double bonds are typically arranged in a linear or cyclic fashion within the molecule. The conjugated system of double bonds is responsible for the absorption of light in the visible spectrum, making carotenoids important pigments in plants, algae, and some microorganisms.

Conjugated Double Bond System:

The fundamental structural feature of carotenoids is the presence of a long series of conjugated double bonds, typically arranged in a polyene chain. This conjugation extends the π-electron cloud along the molecule, resulting in the absorption of light in the visible range and the characteristic coloration exhibited by carotenoids.

Classification of Carotenoids

Carotenoids are classified into two main groups based on their chemical structures and functional groups:

Carotenes:

Carotenes are hydrocarbons, meaning they contain only carbon and hydrogen atoms and lack oxygen-containing functional groups. The absence of oxygen distinguishes carotenes from xanthophylls and contributes to their lipophilic nature. Common examples of carotenes include:

• β-Carotene: A bright orange pigment found in carrots, sweet potatoes, and pumpkins. It is a precursor of vitamin A and serves as a potent antioxidant.
• α-Carotene: Similar in structure to β-carotene but with one less β-ionone ring. It is abundant in green leafy vegetables and contributes to their yellow-orange coloration.
• Lycopene: A red pigment abundant in tomatoes, watermelons, and pink grapefruits. Lycopene lacks β-ionone rings and is highly effective in scavenging free radicals.

Xanthophylls:

Xanthophylls contain oxygen-containing functional groups, such as hydroxyl (-OH) or epoxy (-O-) groups, in addition to their conjugated double bonds. The presence of oxygen atoms imparts unique chemical properties and biological functions to xanthophylls. Common examples of xanthophylls include:

• Lutein: Found in green leafy vegetables, egg yolks, and marigold flowers, lutein is essential for eye health and is concentrated in the macula of the retina.
• Zeaxanthin: Often found alongside lutein in corn, spinach, and orange peppers, zeaxanthin is another important carotenoid for maintaining vision and protecting against age-related macular degeneration.
• Astaxanthin: A red pigment found in seafood such as salmon, shrimp, and lobster. Astaxanthin exhibits powerful antioxidant properties and is responsible for the pink coloration of flamingos and crustaceans.

Sources of Carotenoids

Plants: Carotenoids are predominantly synthesized by plants through a series of enzymatic reactions occurring in plastids, the organelles responsible for photosynthesis. These pigments impart vibrant colors to fruits, vegetables, and flowers, serving as visual cues to attract pollinators and seed dispersers. Common dietary sources of carotenoids include carrots, tomatoes, spinach, and peppers.

Algae: Various species of microalgae and macroalgae also produce carotenoids as part of their photosynthetic machinery. These pigments contribute to the diverse colors observed in aquatic ecosystems and are essential for light harvesting and photoprotection in algae.

Bacteria: Certain photosynthetic bacteria, such as cyanobacteria and purple bacteria, synthesize carotenoids to protect their photosynthetic apparatus from photodamage and oxidative stress. Carotenoids also play a role in regulating the light-harvesting complexes in these organisms.

Fungi: While fungi are unable to synthesize carotenoids de novo, some species can acquire carotenoids from their environment through dietary sources or symbiotic relationships with other organisms. Fungal carotenoids contribute to the coloration of fungal fruiting bodies and play a role in defense against oxidative stress.

Biosynthesis of Carotenoids

The biosynthesis of carotenoids is a complex and highly regulated process that occurs in plastids, particularly chloroplasts in plants and algae. The pathway involves a series of enzymatic reactions that convert simple precursors into a diverse array of carotenoid molecules. The key steps in carotenoid biosynthesis include:

• Formation of Isoprenoid Precursors: The biosynthesis of carotenoids begins with the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which serve as the building blocks for all isoprenoid compounds, including carotenoids. These precursors are synthesized via the methylerythritol phosphate (MEP) pathway or the mevalonate (MVA) pathway, depending on the organism.
• Condensation of Isoprene Units: IPP and DMAPP molecules undergo condensation reactions catalyzed by prenyltransferases to form longer-chain isoprenoid intermediates, such as geranylgeranyl diphosphate (GGPP).
• Formation of Phytoene: The first committed step in carotenoid biosynthesis is the condensation of two molecules of GGPP to form phytoene, a colorless C40 hydrocarbon. This reaction is catalyzed by the enzyme phytoene synthase.
• Desaturation and Cyclization: Phytoene undergoes a series of desaturation and cyclization reactions mediated by desaturases and cyclases, respectively, leading to the formation of colored carotenoid molecules such as lycopene, beta-carotene, and alpha-carotene.
• Diversification and Modification: Further enzymatic reactions, including hydroxylation, epoxidation, and cleavage, introduce additional functional groups and structural modifications to generate a wide range of carotenoid derivatives with distinct biological activities.
• Regulation of Biosynthetic Pathway: The biosynthesis of carotenoids is tightly regulated at the transcriptional, translational, and post-translational levels in response to environmental cues, developmental signals, and metabolic demands. Key regulatory factors include transcription factors, enzyme feedback inhibition, and signaling molecules.

Functions of Carotenoids

Carotenoids are a diverse group of pigments found in plants, animals, and microorganisms, each with unique functions and roles in biological systems. These functions are essential for the health, survival, and ecological interactions of organisms across various habitats.

Antioxidant Activity:

Carotenoids serve as potent antioxidants, neutralizing harmful reactive oxygen species (ROS) and protecting cells from oxidative damage. By donating electrons or hydrogen atoms to free radicals, carotenoids prevent lipid peroxidation, protein oxidation, and DNA damage, thereby maintaining cellular integrity and function.

Photoprotection:

In photosynthetic organisms, such as plants, algae, and cyanobacteria, carotenoids play a crucial role in photoprotection. By dissipating excess light energy and quenching singlet oxygen and other reactive intermediates generated during photosynthesis, carotenoids prevent photodamage to chlorophyll molecules and maintain photosynthetic efficiency.

Light Harvesting:

Carotenoids absorb light energy in the blue and green regions of the spectrum, complementing the absorption of chlorophyll molecules. This expanded absorption range enhances the efficiency of photosynthesis, maximizing energy capture from sunlight and promoting plant growth and productivity.

Pigmentation:

Carotenoids contribute to the vibrant colors of fruits, flowers, and leaves, attracting pollinators and seed dispersers. The diversity of carotenoid pigments enables organisms to produce a spectrum of colors, ranging from yellow and orange to red and purple, enhancing their reproductive success and ecological interactions.

Vision:

In animals, certain carotenoids accumulate in the retina of the eye, where they contribute to visual acuity and protect against age-related macular degeneration (AMD). By absorbing harmful blue light and reducing oxidative stress in retinal cells, carotenoids support healthy vision and maintain ocular health throughout life.

Immune Modulation:

Carotenoids exhibit immunomodulatory effects in animals, enhancing immune responses and promoting resistance to infections and diseases. By regulating inflammatory pathways, modulating cell signaling, and enhancing antioxidant defenses, carotenoids support the body's immune system and contribute to overall health and well-being.

Reproduction:

In many animal species, carotenoid-based coloration serves as an honest indicator of health and reproductive fitness. Brightly colored ornaments, such as feathers, skin patches, or plumage, signal an individual's ability to obtain and metabolize carotenoids, influencing mate choice and reproductive success.

Chemical Defense:

In addition to their roles in physiology and reproduction, carotenoids contribute to chemical defense mechanisms in organisms. By acting as precursors to apocarotenoids, which possess antimicrobial, antifungal, and cytotoxic properties, carotenoids help organisms defend against pathogens, parasites, and predators.

What Does Carotenoids Do For the Body?

• Antioxidants: Carotenoids, such as beta-carotene and lycopene, act as antioxidants in the body, protecting cells from damage caused by harmful free radicals.
• Boost Immune System: They enhance the body's immune system and protect skin from sun damage.
• Improve eye health: Some types of carotenoids, like lutein and zeaxanthin, are concentrated in the eyes where they protect against harmful light waves and may include the risk of age-related macular degeneration and cataracts.
• Reduce risk of cancer and chronic diseases: Carotenoids may reduce the risk of certain types of cancers and chronic diseases, like heart disease, by preventing oxidative damage.
• Improve skin health: Carotenoids can be thought of as nature's sunscreen; when consumed, they are transported to the skin's surface and help to protect the skin against UV light damage.

What are The Methods of Analysis of Carotenoids?

Accurate and reliable analysis of carotenoids is essential for understanding their presence, composition, and concentration in various samples. A variety of analytical techniques are employed to quantify and characterize carotenoids, each with its advantages and limitations.

Characteristic MS and UV–Vis spectra of the main carotenoid compound detected in the saponified extract from Planococcus sp. CP5-4. A MS spectrum, UV–Vis spectrum, and proposed structure for the detected carotenoid compound; and B CID fragmentation pattern of methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate (Moyo et al., 2022).

High-Performance Liquid Chromatography (HPLC):

HPLC is the gold standard technique for carotenoid analysis due to its high sensitivity, resolution, and versatility. In HPLC analysis, carotenoids are separated based on their chemical properties, such as polarity and molecular size, using a chromatographic column packed with a stationary phase. Detection is typically performed using a UV-Vis detector or a photodiode array detector, allowing for quantification of individual carotenoids based on their retention times and absorbance spectra. HPLC is suitable for analyzing complex mixtures of carotenoids in various sample matrices, including foods, biological tissues, and dietary supplements.

Gas Chromatography (GC):

GC is another widely used technique for carotenoid analysis, particularly for volatile and semi-volatile compounds. In GC analysis, carotenoids are converted into volatile derivatives, such as silyl ethers or esters, before injection onto a chromatographic column. Separation is achieved based on the compounds' vapor pressures and affinity for the stationary phase. Detection is typically performed using a flame ionization detector (FID) or a mass spectrometer (MS), enabling quantification of individual carotenoids based on their retention times and mass spectra. GC is well-suited for analyzing carotenoids in lipid-rich samples, such as oils, fats, and waxes.

UV-Visible Spectrophotometry:

UV-Visible spectrophotometry is a simple and cost-effective method for quantifying total carotenoid content in samples. In this method, carotenoids are extracted from the sample using organic solvents, such as acetone or ethanol, and their absorbance is measured at specific wavelengths in the UV-Vis range. The absorbance values are then correlated with carotenoid concentrations using calibration curves generated from standard solutions. While UV-Visible spectrophotometry provides a rapid screening tool for estimating carotenoid content, it lacks specificity and cannot differentiate between individual carotenoids.

Thin-Layer Chromatography (TLC):

TLC is a qualitative technique used for separating and identifying different carotenoids in complex mixtures. In TLC analysis, a small amount of the sample is spotted onto a thin layer of adsorbent material, such as silica gel or cellulose, on a glass or plastic plate. The plate is then placed in a developing chamber containing a solvent system, which migrates up the plate via capillary action, separating the carotenoids based on their affinity for the stationary phase and the mobile phase. After development, the separated compounds are visualized using chemical reagents or under UV light. TLC is valuable for preliminary screening of carotenoids but lacks the sensitivity and precision of other analytical techniques.

Mass Spectrometry (MS):

MS is a powerful technique for identifying and quantifying carotenoids based on their mass-to-charge ratios and fragmentation patterns. In MS analysis, carotenoids are ionized and fragmented using techniques such as electron ionization (EI) or electrospray ionization (ESI), and the resulting ions are analyzed using a mass analyzer. MS can provide structural information about carotenoids, such as molecular weight, double bond positions, and functional group substitutions, facilitating their identification and characterization. Coupling MS with chromatographic techniques, such as HPLC or GC, allows for comprehensive analysis of carotenoid mixtures in complex samples.

References

1. Ishida, B. K., and G. E. Bartley. "Carotenoids: chemistry, sources, and physiology." Encyclopedia of human nutrition 1 (2005): 330-338.
2. Moyo, Anesu Conrad, et al. "Structure and biosynthesis of carotenoids produced by a novel Planococcus sp. isolated from South Africa." Microbial Cell Factories 21.1 (2022): 43.