Vitamin: Metabolism, Functions, Analytical Methods and Applications

What are Vitamins?

Vitamins are organic compounds that are necessary for the proper functioning of metabolic pathways. They are classified as essential nutrients because the human body cannot synthesize them in adequate amounts, necessitating their intake through diet or supplementation. Each vitamin serves a distinct biochemical role, often acting as a coenzyme or a precursor in metabolic reactions.

Key characteristics of vitamins include:

• Essential for normal cellular functions and metabolism
• Required in small quantities
• Deficiency can lead to specific diseases or metabolic disorders
• Do not provide energy but facilitate energy production

Classification of Vitamins

Vitamins are broadly categorized based on their solubility properties, which influence their absorption, storage, and excretion:

Water-Soluble Vitamins

Water-soluble vitamins dissolve in water and are readily excreted in urine, leading to a lower risk of toxicity compared to fat-soluble vitamins. However, they require continuous dietary intake to maintain adequate levels.

• Vitamin B Complex: Includes B1 (Thiamine), B2 (Riboflavin), B3 (Niacin), B5 (Pantothenic Acid), B6 (Pyridoxine), B7 (Biotin), B9 (Folate), and B12 (Cobalamin). They primarily act as coenzymes in energy metabolism.
• Vitamin C (Ascorbic Acid): Functions as a powerful antioxidant, supporting collagen synthesis, iron absorption, and immune defense.

Fat-Soluble Vitamins

Fat-soluble vitamins are absorbed alongside dietary fats and stored in adipose tissues and the liver. Their excessive accumulation can lead to toxicity.

• Vitamin A: Essential for vision, immune function, and cellular growth.
• Vitamin D: Regulates calcium and phosphorus metabolism, crucial for bone health.
• Vitamin E: Acts as an antioxidant, protecting cell membranes from oxidative damage.
• Vitamin K: Plays a key role in blood clotting and bone metabolism.

Vitamin Metabolism

Vitamin metabolism encompasses a series of biochemical processes through which vitamins are absorbed, transported, transformed into active forms, utilized in physiological functions, and ultimately excreted. Each vitamin follows distinct metabolic pathways depending on its chemical nature, with regulation at multiple levels to maintain homeostasis.

Absorption and Transport of Vitamin

The absorption of vitamins primarily occurs in the small intestine, with different mechanisms employed for water-soluble and fat-soluble vitamins.

• Water-Soluble Vitamins: These vitamins, including all B-complex vitamins and vitamin C, are absorbed through active transport or facilitated diffusion. Specialized carrier proteins, such as sodium-dependent vitamin transporters (e.g., SVCT1 for vitamin C and THTR1 for thiamine), mediate uptake into enterocytes. In cases of excess intake, passive diffusion may contribute to absorption.
• Fat-Soluble Vitamins: Vitamins A, D, E, and K require the presence of dietary fats and bile salts for solubilization into micelles. Once absorbed into enterocytes, they are incorporated into chylomicrons and transported through the lymphatic system before entering the bloodstream.

Activation and Biochemical Conversion

Many vitamins undergo biochemical transformations to become biologically active. This activation often involves phosphorylation, hydroxylation, or methylation.

• Vitamin D: After intestinal absorption, vitamin D3 (cholecalciferol) is transported to the liver, where it undergoes 25-hydroxylation by the enzyme CYP2R1 to form 25-hydroxyvitamin D [25(OH)D]. Subsequently, in the kidneys, CYP27B1 catalyzes the conversion of 25(OH)D to its active form, 1,25-dihydroxyvitamin D [1,25(OH)2D], which regulates calcium and phosphate homeostasis.
• Vitamin B6: Pyridoxine, pyridoxal, and pyridoxamine are phosphorylated by pyridoxal kinase to produce pyridoxal 5'-phosphate (PLP), the active coenzyme form. PLP participates in amino acid metabolism through transamination, decarboxylation, and deamination reactions.
• Vitamin K: Vitamin K is essential for post-translational γ-carboxylation of glutamic acid residues in clotting factors. The reduced form of vitamin K, hydroquinone (KH2), serves as a cofactor in this carboxylation reaction, which is catalyzed by the enzyme γ-glutamyl carboxylase.

Schematic overview of vitamin D metabolism in humans (Spyksma, Eva E., et al., 2024).

Cellular Uptake and Utilization

Once in circulation, vitamins are taken up by specific tissues where they perform their biological functions.

• Cellular Transport Mechanisms: Water-soluble vitamins typically rely on membrane transporters, such as the reduced folate carrier (RFC) for folate or the transcobalamin receptor for vitamin B12. Fat-soluble vitamins are delivered to cells via lipoproteins like low-density lipoproteins (LDL) or high-density lipoproteins (HDL).
• Intracellular Activation: In cells, further modifications may occur. For example, riboflavin (vitamin B2) is phosphorylated to flavin mononucleotide (FMN) and subsequently to flavin adenine dinucleotide (FAD), both essential cofactors in redox reactions.

Regulation of Vitamin Metabolism

Vitamin metabolism is tightly regulated through feedback mechanisms and hormonal control to ensure physiological balance.

• Calcium-Phosphate Homeostasis: The active form of vitamin D, 1,25(OH)2D, increases intestinal calcium absorption. When calcium levels are sufficient, parathyroid hormone (PTH) secretion decreases, reducing further conversion of 25(OH)D to its active form.
• Redox Balance: Vitamins with antioxidant properties, such as vitamins C and E, participate in the regulation of oxidative stress. Oxidized vitamin E can be regenerated by vitamin C, maintaining antioxidant capacity.
• Enzymatic Regulation: Several vitamins act as allosteric regulators of enzymes. For instance, thiamine pyrophosphate (TPP), the active form of vitamin B1, regulates the pyruvate dehydrogenase complex, linking glycolysis to the citric acid cycle.

Storage and Mobilization

The storage capacity of vitamins varies depending on their solubility.

• Fat-Soluble Vitamins: Significant amounts of vitamins A, D, E, and K are stored in the liver and adipose tissue. Vitamin A, in particular, is stored as retinyl esters in hepatic stellate cells. These reservoirs can supply the body with vitamins for extended periods during dietary deficiency.
• Water-Soluble Vitamins: With the exception of vitamin B12, which can be stored in the liver for years, water-soluble vitamins are not stored in large amounts. Continuous intake is necessary to prevent depletion.

Excretion

Excretion pathways differ between water-soluble and fat-soluble vitamins.

• Water-Soluble Vitamins: Excess water-soluble vitamins are typically excreted through the kidneys into the urine. Renal reabsorption mechanisms may reclaim some vitamins, especially in states of deficiency.
• Fat-Soluble Vitamins: These vitamins are primarily excreted through the feces via biliary secretion. The enterohepatic circulation allows for the recycling of fat-soluble vitamins, reducing their loss.

Functions and Roles of Vitamins

Coenzyme and Cofactor Functions

Many vitamins serve as coenzymes or precursors for coenzymes, facilitating enzymatic reactions essential for energy production, macronutrient metabolism, and biosynthesis.

• B-Complex Vitamins:
  • Thiamine (Vitamin B1) functions as thiamine pyrophosphate (TPP), a crucial coenzyme in the decarboxylation of α-keto acids during glycolysis and the citric acid cycle.
  • Riboflavin (Vitamin B2) forms flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), both acting as electron carriers in redox reactions within the mitochondrial electron transport chain.
  • Niacin (Vitamin B3) is a precursor of nicotinamide adenine dinucleotide (NAD) and its phosphate form (NADP), essential for oxidative and reductive metabolism.
  • Biotin (Vitamin B7) serves as a coenzyme for carboxylase enzymes in fatty acid synthesis, gluconeogenesis, and amino acid metabolism.
• Vitamin C: While not a typical coenzyme, ascorbic acid acts as a reducing agent in hydroxylation reactions, particularly in collagen synthesis. It maintains iron in its ferrous state (Fe²⁺) for optimal enzymatic activity.

Antioxidant Defense and Redox Regulation

Certain vitamins act as antioxidants, neutralizing reactive oxygen species (ROS) and preventing oxidative damage to lipids, proteins, and DNA.

• Vitamin E (Tocopherol): Embedded in cell membranes, vitamin E protects polyunsaturated fatty acids (PUFAs) from lipid peroxidation. It donates electrons to stabilize free radicals, interrupting the chain reaction of oxidative damage.
• Vitamin C (Ascorbic Acid): Besides its role in collagen synthesis, vitamin C regenerates oxidized vitamin E and scavenges ROS in aqueous environments.
• Vitamin A (Retinoids): As a lipid-soluble antioxidant, retinoids mitigate oxidative stress in tissues, particularly in photoreceptor cells of the retina.

Gene Expression and Cellular Regulation

Some vitamins directly influence gene expression and cellular differentiation.

• Vitamin A (Retinoic Acid): The active form of vitamin A binds to nuclear receptors, such as retinoic acid receptors (RARs) and retinoid X receptors (RXRs), regulating gene transcription. This controls cellular differentiation, proliferation, and apoptosis, especially in epithelial tissues.
• Vitamin D (Calcitriol): Acting as a steroid hormone, calcitriol binds to the vitamin D receptor (VDR), forming a transcriptional complex that regulates genes involved in calcium homeostasis, bone metabolism, and immune function.

Immune Modulation and Inflammation Control

Vitamins play vital roles in the immune response, influencing both innate and adaptive immunity.

• Vitamin D: Regulates the expression of antimicrobial peptides like cathelicidin and defensins. It also modulates cytokine production and suppresses excessive inflammation, reducing the risk of autoimmune diseases.
• Vitamin A: Supports mucosal immunity by maintaining epithelial barrier integrity and promoting immunoglobulin A (IgA) secretion. It also enhances the differentiation of T cells, particularly regulatory T cells (Tregs).
• Vitamin C and Vitamin E: Both vitamins mitigate inflammation by neutralizing ROS and inhibiting pro-inflammatory cytokines.

Hemostasis and Blood Coagulation

Vitamin K: Vitamin K serves as a cofactor for γ-glutamyl carboxylase, which catalyzes the carboxylation of glutamate residues in clotting factors II, VII, IX, and X. This post-translational modification is essential for their biological activity in the coagulation cascade. In addition, vitamin K supports bone health by activating osteocalcin, a protein involved in bone mineralization.

Bone and Mineral Homeostasis

• Vitamin D: By regulating calcium and phosphate absorption in the intestines, vitamin D ensures proper bone mineralization. It stimulates the release of calcium from bones when necessary, maintaining serum calcium levels within a narrow range.
• Vitamin K: In addition to its coagulation role, vitamin K contributes to bone metabolism through its involvement in osteocalcin activation.

Neurological and Cognitive Functions

Vitamins are indispensable for maintaining nervous system function, neurotransmitter synthesis, and myelin integrity.

• Vitamin B1 (Thiamine): Thiamine deficiency can lead to Wernicke-Korsakoff syndrome, characterized by neurological and cognitive impairments. TPP-dependent enzymes are crucial in glucose metabolism, providing energy for neurons.
• Vitamin B6 (Pyridoxine): Pyridoxal phosphate (PLP) is a coenzyme in neurotransmitter synthesis, including serotonin, dopamine, and gamma-aminobutyric acid (GABA).
• Vitamin B12 (Cobalamin) and Folate (Vitamin B9): Both vitamins are essential for DNA synthesis and methylation, supporting neuronal health and preventing neurodegenerative diseases. Deficiencies can result in peripheral neuropathy and cognitive decline.

Reproductive and Fetal Development

• Folate (Vitamin B9): Folate is critical during embryogenesis for neural tube closure. Deficiency can lead to neural tube defects such as spina bifida and anencephaly.
• Vitamin A: Retinoic acid regulates gene expression during embryonic development, guiding proper organogenesis.
• Vitamin D: Sufficient maternal vitamin D levels are essential for fetal skeletal development and immune function.

Energy Production and Metabolism

• B-Complex Vitamins: These vitamins are key regulators of energy metabolism. For example, vitamin B3 (niacin) and vitamin B2 (riboflavin) facilitate oxidative phosphorylation by participating in the electron transport chain.
• Biotin (Vitamin B7): Acts as a coenzyme in carboxylation reactions, particularly in gluconeogenesis, fatty acid synthesis, and amino acid catabolism.

A diagram illustrating the roles of various vitamins on different organ systems (Manna, Pulak R., et al., 2022).

How to Analyze Vitamins?

Sample Preparation

Effective sample preparation is essential to extract vitamins from complex matrices without degradation or loss. Different methods are applied depending on whether the sample is biological (e.g., plasma, urine, tissue) or food-based.

• Extraction: Fat-soluble vitamins (A, D, E, K) often require organic solvent extraction using hexane, chloroform, or ethanol, while water-soluble vitamins (B-complex, C) are extracted using aqueous solutions or acidic buffers.
• Protein Precipitation: Biological fluids are often treated with organic solvents like acetonitrile or methanol to precipitate proteins and release vitamins for analysis.
• Derivatization: Some vitamins, such as thiamine (B1) and pyridoxine (B6), require derivatization with reagents like benzoyl chloride or o-phthaldialdehyde (OPA) to enhance their detectability during chromatographic analysis.

Chromatographic Techniques

Chromatography remains the gold standard for vitamin analysis due to its precision, sensitivity, and capability to separate vitamins from complex mixtures.

High-Performance Liquid Chromatography (HPLC):

• Reversed-Phase HPLC (RP-HPLC) is commonly used for both fat-soluble and water-soluble vitamins using C18 columns and UV or fluorescence detectors.
• Ion-Pair HPLC is advantageous for the separation of ionic vitamins like vitamin C and certain B-complex vitamins.
• Ultra-Performance Liquid Chromatography (UPLC) offers higher resolution and faster analysis than traditional HPLC.

Gas Chromatography (GC):

Suitable for volatile or derivatized vitamins like vitamin E (tocopherols) and vitamin D metabolites. GC coupled with mass spectrometry (GC-MS) provides high sensitivity and specificity.

Thin-Layer Chromatography (TLC):

Although less sensitive, TLC remains a cost-effective option for qualitative vitamin analysis, often used in preliminary assessments.

Mass Spectrometry (MS)

Mass spectrometry is widely applied for the precise quantification of vitamins and their metabolites. It is often coupled with chromatography for enhanced specificity.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS):

LC-MS/MS is the preferred method for detecting vitamins in biological samples due to its ability to quantify multiple analytes in a single run. It is particularly effective for analyzing vitamin D metabolites, B vitamins, and retinoids.

Gas Chromatography-Mass Spectrometry (GC-MS):

Used for the analysis of fat-soluble vitamins and their oxidation products, GC-MS is effective in detecting lipid-soluble vitamin derivatives in plasma and tissues.

Spectrophotometric and Fluorometric Methods

UV-Vis Spectrophotometry:

Vitamins with characteristic UV absorption, such as vitamin A (retinol) and vitamin C (ascorbic acid), can be quantified using UV-Vis spectrophotometry. However, it may lack specificity when interfering substances are present.

Fluorometry:

Some vitamins, like riboflavin (B2) and its derivatives, exhibit natural fluorescence, enabling sensitive detection using fluorometers. Pre-column or post-column derivatization can enhance fluorescence for vitamins like thiamine.

Immunoassays and Biosensors

Enzyme-Linked Immunosorbent Assay (ELISA):

ELISA is often used for detecting vitamins in biological samples. It is particularly applied in large-scale clinical studies due to its simplicity and high-throughput capability.

Biosensors:

Recent advancements in biosensor technology provide rapid and real-time vitamin detection using enzyme-based or nanoparticle-based sensors. These are valuable in point-of-care testing and food quality monitoring.

Isotopic and Radioactive Methods

Isotope Dilution Mass Spectrometry (IDMS):

IDMS is a highly accurate and precise method that involves spiking samples with stable isotope-labeled vitamins. This technique is commonly applied in reference laboratories for vitamin D and folate analysis.

Radioimmunoassay (RIA):

Though less frequently used today, RIA remains a sensitive method for analyzing trace levels of vitamins, particularly in endocrinological studies.

Quality Control and Method Validation

For reliable vitamin analysis, analytical methods must undergo rigorous validation. This includes assessing parameters such as linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and stability. Certified reference materials (CRMs) and participation in external quality assessment programs further ensure the accuracy and comparability of results.

• Inter-Laboratory Comparisons: Proficiency testing through standardized programs is critical for ensuring consistent and accurate vitamin analysis across different laboratories.
• Calibration and Internal Standards: Internal standards, often stable isotope-labeled vitamins, are used to correct for matrix effects and variability in analytical procedures.

Applications of Vitamin Analysis

Food Industry and Nutritional Fortification

Accurate vitamin quantification ensures compliance with nutritional labeling and fortification programs. For instance, LC-MS/MS detects fat-soluble vitamins (A, D, E, K) in fortified dairy products, addressing deficiencies like rickets and supporting global public health initiatives. HPLC-UV methods validate vitamin C stability in juices and supplements, where degradation due to heat or light impacts bioavailability. Emerging applications include functional foods targeting specific demographics (e.g., vitamin D-enriched cereals for children), with microbial fermentation enabling cost-effective production of B-complex vitamins for mass fortification.

Agricultural and Feed Optimization

In livestock nutrition, vitamin analysis optimizes feed formulations to enhance animal health and productivity. For example, vitamin E (α-tocopherol) supplementation reduces oxidative stress in poultry, improving meat quality and reproductive performance. LC-MS/MS quantifies vitamin K2 (menaquinones) in feed additives, which support bone development in poultry and dairy cattle. Microbial engineering further enhances feed-grade vitamin production—Bacillus subtilis strains engineered via adaptive laboratory evolution (ALE) achieve 300% yield increases in menaquinone-7 synthesis, reducing reliance on synthetic methods.

Cosmetic Formulation and Stability Testing

Vitamins are integral to anti-aging and protective skincare formulations. Retinol (vitamin A) derivatives require precise stability testing via HPLC-UV to prevent degradation in creams and serums. Encapsulation technologies (e.g., liposomes) extend shelf life by shielding vitamin C (ascorbic acid) from oxidation, ensuring sustained efficacy in brightening products. Analytical methods also validate synergistic combinations, such as vitamin E with ferulic acid, which amplifies antioxidant capacity in sunscreens.

Industrial Biotechnology and Metabolic Engineering

Vitamin analysis drives microbial cell factory development. Escherichia coli engineered for vitamin B12 production requires LC-MS/MS to monitor intermediates like cobinamide, optimizing methyltransferase activity. Similarly, CRISPR-edited Saccharomyces cerevisiae strains producing β-carotene (a vitamin A precursor) are validated via HPLC, enabling scalable synthesis for food and cosmetic industries. Dynamic flux analysis identifies bottlenecks in microbial pathways, such as NADPH dependency in riboflavin (B2) biosynthesis, guiding metabolic engineering strategies.

Environmental Monitoring

Vitamin analysis tracks environmental contaminants. For example, vitamin K1 in water systems serves as a biomarker for agricultural runoff, reflecting fertilizer use and ecosystem health. LC-MS/MS detects trace levels (ng/L) in aquatic samples, aiding pollution mitigation strategies.

References

1. Spyksma, Eva E., et al. "An overview of different vitamin D compounds in the setting of adiposity." Nutrients 16.2 (2024): 231. https://doi.org/10.3390/nu16020231
2. Manna, Pulak R., Zackery C. Gray, and P. Hemachandra Reddy. "Healthy immunity on preventive medicine for combating COVID-19." Nutrients 14.5 (2022): 1004. https://doi.org/10.3390/nu14051004
3. Delompré, Thomas, et al. "Taste perception of nutrients found in nutritional supplements: A review." Nutrients 11.9 (2019): 2050. https://doi.org/10.3390/nu11092050
4. Köseoğlu, Kadir, et al. "Simple and sensitive determination of vitamin A and E in the milk and egg yolk samples by using dispersive solid phase extraction with newly synthesized polymeric material." Journal of Food Composition and Analysis 90 (2020): 103482. https://doi.org/10.1016/j.jfca.2020.103482