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Hydroxyproline

Hydroxyproline is a non-proteinogenic α-imino acid that serves as a post-translationally modified derivative of proline, distinguished by the addition of a hydroxyl group to its pyrrolidine ring.^[1] It is not incorporated directly into proteins during translation but is formed enzymatically within specific polypeptides, most notably collagen, where glycine, proline, and hydroxyproline together constitute approximately 57% of the amino acid residues.^[2] This modification is essential for the structural integrity and stability of collagen, the most abundant protein in animals, which accounts for about one-third of total body protein.^[2] Chemically, hydroxyproline exists primarily as (2S,4R)-4-hydroxyproline, featuring a hydroxyl group at the 4-position of the proline ring, which imparts rigidity and facilitates hydrogen bonding critical for the triple-helical conformation of collagen.^[1] Biosynthesis occurs via hydroxylation of proline residues in nascent procollagen chains, catalyzed by prolyl 4-hydroxylase (or prolyl 3-hydroxylase for the 3-hydroxy variant), an enzyme that requires molecular oxygen, ascorbic acid (vitamin C), α-ketoglutarate, and ferrous iron as cofactors.^[1] This process is oxygen-dependent and occurs in the endoplasmic reticulum, ensuring proper folding and secretion of collagen to the extracellular matrix; deficiencies, such as in scurvy, impair hydroxylation and lead to collagen instability.^[3] Beyond collagen, hydroxyproline contributes to various biological functions, including redox regulation by scavenging reactive oxygen species and serving as a metabolic intermediate in the degradation of connective tissues.^[1] In metabolism, it is catabolized primarily in the liver to yield glycine, pyruvate, and ultimately glucose, with urinary excretion of excess amounts often used as a biomarker for collagen turnover in conditions like fibrosis or bone disorders.^[1] Dietary hydroxyproline, derived from collagen-rich foods, supports growth in neonates and certain animals but is conditionally essential in humans due to its reliance on endogenous synthesis.^[2] Its presence in other proteins and as an osmolyte in some organisms underscores its broader evolutionary significance in structural and adaptive biology.^[1]

Chemical Properties

Molecular Structure

Hydroxyproline, specifically the predominant form trans-4-hydroxy-L-proline, has the molecular formula \ce{C5H9NO3} and is classified as a modified amino acid.^[4] It is a cyclic imino acid derived from proline, featuring a five-membered pyrrolidine ring with a hydroxyl group substituted at the 4-position in the trans configuration relative to the carboxylic acid group at the 2-position.^[4] The systematic IUPAC name is (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid, where the stereochemistry at C2 (S) and C4 (R) defines the L-trans chirality, distinguishing it from other isomers and influencing its rigidity and hydrogen-bonding potential in proteins.^[4] The molecular weight of trans-4-hydroxy-L-proline is 131.13 g/mol.^[4] It exhibits high solubility in water, approximately 361 mg/mL at 25°C, due to its polar hydroxyl and charged groups, while being slightly soluble in ethanol and insoluble in ether.^[4] The pKa values are approximately 1.82 for the carboxyl group and 9.65 for the amino group, reflecting typical acidic and basic dissociation behaviors of amino acids that enable its zwitterionic form under physiological conditions.^[4] Spectroscopic methods are essential for identifying and characterizing hydroxyproline in biochemical samples. In nuclear magnetic resonance (NMR) spectroscopy, the 1H NMR spectrum (500 MHz, H2O, pH 7) shows characteristic proton shifts at δ 4.33 (H-2), 4.67 (H-4), 2.14 and 2.42 (H-3), 3.36 and 3.46 (H-5), and ~3.36 ppm (OH, exchangeable; may overlap with H-5 signal), highlighting the ring protons and hydroxyl.^[4] The 13C NMR spectrum (125 MHz, H2O, pH 7) displays shifts at δ 176.93 (C=O), 72.80 (C-4), 62.62 (C-2), 55.75 and 40.12 ppm (C-3 and C-5), confirming the substituted pyrrolidine structure.^[4] Infrared (IR) spectroscopy reveals key absorptions for the hydroxyl (around 3400 cm⁻¹, broad O-H stretch), carbonyl (approximately 1700 cm⁻¹, C=O), and amine groups, as seen in FTIR spectra of the compound, aiding in structural verification.^[4]

Isomers and Derivatives

Hydroxyproline exists in multiple isomeric forms due to the position of the hydroxyl group and stereochemical configurations at the alpha carbon (C2) and the hydroxyl-bearing carbon. The primary naturally occurring isomer is trans-4-hydroxy-L-proline, designated as (2S,4R)-4-hydroxyproline, where the hydroxyl group at the 4-position is trans to the carboxylic acid group. In contrast, the cis-4-hydroxy-L-proline isomer, (2S,4S)-4-hydroxyproline, features the hydroxyl group cis to the carboxylic acid, resulting in a more puckered pyrrolidine ring conformation that influences peptide rigidity. These stereoisomers differ in their ring pucker and hydrogen-bonding potential, with the trans form predominant in most biological contexts.^[5]^[6] Another key isomer is 3-hydroxy-L-proline, which bears the hydroxyl at the 3-position instead of the 4-position, yielding trans-(2S,3S)-3-hydroxyproline and cis-(2S,3R)-3-hydroxyproline variants. This isomer is rarer and occurs primarily in specific collagens, such as type IV collagen in basement membranes, where it constitutes about 6 to 16 residues per 1,000 amino acids, contributing to unique structural modifications. Unlike the more common 4-hydroxyproline, 3-hydroxyproline's presence is limited to sites like the Gly-3-Hyp-4-Hyp triplet in collagen alpha chains.^[7]^[8] Derivatives of hydroxyproline often involve further modifications, such as glycosylation, which attach carbohydrate moieties to the hydroxyl group. A prominent example is O-arabinosylated hydroxyproline, found in plant cell wall extensins, where a β-L-arabinofuranose unit links to the 4-hydroxyl of trans-4-hydroxyproline, forming structures like Hyp-(β-Ara)f that promote cross-linking in glycoproteins. In animal systems, hydroxyproline-containing peptides, such as those derived from collagen hydrolysis, can undergo O-glycosylation with galactose or glucosylgalactose, enhancing solubility and stability; for instance, the disaccharide Glc(β1→2)Gal(β1→O)-Hyp occurs in basement membrane collagens. These derivatives are represented chemically as the pyrrolidine ring of proline with a -OH at C4 substituted by -O-CH₂-(CHOH)₃-CH₂OH for simple arabinosides, though polymeric forms extend this motif.^[9]^[10]^[11] Synthetic production of hydroxyproline isomers employs stereoselective methods to access specific configurations. Enzymatic approaches utilize proline hydroxylases, such as P4H for trans-4-hydroxyproline or P3H for 3-hydroxyproline, enabling regio- and stereoselective hydroxylation of L-proline with high yields (up to 95% ee) under mild conditions like pH 7 and 25°C. Chemical syntheses include asymmetric dihydroxylation or chiral auxiliary-mediated reactions; for example, the four stereoisomers of 4-hydroxyproline can be prepared from furan derivatives via stereocontrolled epoxidation, achieving diastereoselectivities >20:1. These methods allow scalable production for pharmaceutical applications, such as in proline mimetics.^[12]^[13]^[14]

History and Discovery

Early Identification

Hydroxyproline was first isolated in 1902 by German chemist Hermann Emil Fischer through acid hydrolysis of gelatin, a collagen-derived protein from animal connective tissues.^[15] This discovery marked the initial recognition of hydroxyproline as a unique imino acid component abundant in animal collagen, distinguishing it from common amino acids like proline. Fischer's work involved fractionating the hydrolysate and identifying the compound based on its chemical properties, including its crystalline form and reactions typical of hydroxylated pyrrolidine derivatives. Early experiments focused on hydrolysis of gelatin to yield hydroxyproline-rich fractions, confirming its prevalence in collagenous materials. In the 1930s, researchers began emphasizing its role as a hallmark of collagen structure, with quantitative analyses revealing it constituted approximately 13-14% of collagen's amino acid content by weight. By the 1940s, advancements in quantification methods, such as adaptations of manometric techniques, allowed for more precise measurement of imino acids in biological samples. Notably, in 1948, Donald Van Slyke and colleagues developed a method to determine the combined α-nitrogen of proline and hydroxyproline, enabling better assessment of their contributions to protein hydrolysates from collagen sources.56219-0/fulltext) Initial nomenclature for hydroxyproline reflected confusion with other imino acids due to structural similarities with proline, leading to debates on whether it was a primary amino acid or a modified derivative. This uncertainty persisted until confirmatory syntheses, such as Hermann Leuchs' racemic mixture in 1905, solidified its identity as 4-hydroxyproline.^[15] By 1948, through chromatographic and nitrogen-specific assays, hydroxyproline was firmly established as a distinct residue integral to collagen stability, separate from proline in metabolic and structural contexts.56219-0/fulltext)

Biochemical Characterization

In the 1960s, biochemical studies confirmed that hydroxyproline is formed as a post-translational modification of proline residues in collagen precursors, catalyzed by the enzyme prolyl 4-hydroxylase (P4H), also known as protocollagen proline hydroxylase.^[16] This process was first demonstrated in vitro using microsomal fractions from animal tissues, where proline in nascent polypeptide chains was hydroxylated to yield hydroxyproline, establishing the enzymatic basis for its incorporation into mature collagen.^[17] The reaction requires molecular oxygen, ferrous iron (Fe²⁺), α-ketoglutarate, and ascorbate as essential cofactors, with ascorbate serving to maintain the iron in its reduced state and prevent enzyme inactivation.90716-9) Key investigations by researchers including Prockop and Kivirikko during this period elucidated the enzyme's mechanistic requirements and oxygen dependence. Their work showed that P4H belongs to the family of 2-oxoglutarate-dependent dioxygenases, where α-ketoglutarate and O₂ are stoichiometrically coupled to proline hydroxylation, producing succinate and CO₂ as byproducts.90396-7) These studies highlighted the enzyme's absolute dependence on atmospheric oxygen for activity, linking hydroxyproline formation to cellular oxygen levels and explaining its role in collagen stability under physiological conditions. Ascorbate's cofactor role was particularly emphasized, as its deficiency impairs hydroxylation, leading to underhydroxylated collagen.90716-9) A major milestone occurred in 1965 with the initial isolation of P4H activity from microsomal preparations, enabling detailed enzymatic assays.^[16] Subsequent purification efforts in 1967 yielded highly enriched enzyme from chick embryo extracts, allowing characterization of its molecular properties and substrate specificity for X-Pro-Gly sequences in protocollagen.90396-7) By the 1970s, X-ray crystallographic analyses of synthetic peptides containing hydroxyproline residues provided structural confirmation of its conformational influence, revealing how the 4-hydroxyl group stabilizes the trans peptide bond and promotes the polyproline II helix essential for collagen triple-helix assembly.90175-1)

Biosynthesis

In Animals

In animals, hydroxyproline is primarily synthesized through a post-translational modification of proline residues during the biosynthesis of collagen, the most abundant protein in the extracellular matrix. This process occurs in the endoplasmic reticulum of cells actively producing collagen, such as fibroblasts and osteoblasts, where nascent procollagen chains serve as substrates. The key enzyme responsible is prolyl-4-hydroxylase (P4H), also known as collagen prolyl-4-hydroxylase (C-P4H), a member of the 2-oxoglutarate-dependent dioxygenase family. P4H specifically hydroxylates proline residues at the 4-position within the sequence X-Pro-Gly (where X is often glycine or another amino acid), which is characteristic of collagen's repeating triplet structure. This enzyme exists as an α₂β₂ tetramer, with the α subunit providing catalytic activity and the β subunit (protein disulfide isomerase) aiding in substrate recognition and chain assembly.^[18] The reaction catalyzed by P4H is a stereospecific hydroxylation that incorporates molecular oxygen, requiring iron (Fe²⁺) as a cofactor, ascorbic acid (vitamin C) to maintain the iron in its reduced state, and 2-oxoglutarate as a co-substrate. The overall reaction can be represented as:

\text{Peptidyl-Pro} + 2\text{-oxoglutarate} + \mathrm{O_2} \xrightarrow{\text{P4H, Fe}^{2+}, \text{ascorbate}} \text{Peptidyl-4-hydroxyPro} + \text{succinate} + \mathrm{CO_2}

This uncoupled decarboxylation of 2-oxoglutarate provides the driving force for the hydroxylation, ensuring efficient modification under physiological conditions. The process is oxygen-dependent, linking hydroxyproline formation directly to cellular oxygen availability. Defects in this pathway, such as those caused by ascorbate deficiency, lead to under-hydroxylation of procollagen, impairing its proper folding and secretion.^[19]^[18] Regulation of P4H activity in animals is multifaceted, involving transcriptional control influenced by hypoxia-inducible factor (HIF)-1, which upregulates the expression of the P4H α(I) subunit under low oxygen conditions to modulate collagen synthesis in response to environmental stress. Tissue-specific expression is prominent in collagen-producing cells like fibroblasts, chondrocytes, and osteoblasts, where P4H levels correlate with the demand for extracellular matrix deposition during wound healing, bone formation, and tissue remodeling. Multiple isoenzymes (e.g., types I and II) exist, differing in their α subunit isoforms and contributing to fine-tuned hydroxylation in various tissues. Quantitatively, 4-hydroxyproline constitutes approximately 10-13% of the amino acid residues in mature collagen, highlighting its prevalence and structural importance in animal connective tissues.^[20]^[21]

In Plants and Microorganisms

In plants, hydroxyproline is predominantly incorporated into hydroxyproline-rich glycoproteins (HRGPs), a superfamily of cell wall proteins that includes extensins, arabinogalactan proteins, and proline-rich proteins, which contribute to primary cell wall structure and integrity.^[22] These proteins are synthesized through post-translational hydroxylation of peptidyl proline residues, a process catalyzed by prolyl 4-hydroxylases (P4Hs) in the endoplasmic reticulum, requiring molecular oxygen, 2-oxoglutarate, iron, and ascorbate as cofactors. Unlike the animal pathway focused on collagen modification, plant P4Hs act on diverse proline-rich motifs within HRGPs, leading to subsequent O-glycosylation on hydroxyproline residues that enhances protein insolubility and cross-linking in the cell wall.^[23] In Arabidopsis thaliana, 13 isoforms of P4H (P4H1 through P4H13) have been identified, each exhibiting varying substrate preferences for proline-rich, collagen-like, or hypoxia-responsive peptides, with distinct expression patterns in response to environmental stresses like low oxygen.^[24] Microbial hydroxyproline biosynthesis exhibits key differences from plant and animal pathways, often involving the direct hydroxylation of free L-proline rather than peptidyl forms, resulting in accumulation of free trans-4-hydroxy-L-proline. In bacteria such as Pseudomonas stutzeri and Pseudomonas aeruginosa, this is mediated by trans-proline 4-hydroxylases (trans-P4H), 2-oxoglutarate-dependent dioxygenases that produce hydroxyproline used in secondary metabolites or as a carbon source, with recombinant expression yielding up to 6.72 g/L in optimized strains.^[25] Protists, including green algae like Chlamydomonas reinhardtii, incorporate hydroxyproline into cell wall HRGPs similar to those in higher plants, where these glycoproteins form the entire extracellular matrix and accumulate during cell wall regeneration, regulated by signaling pathways responsive to mechanical stress.^[26] A notable distinction in non-animal systems is the greater diversity of hydroxyproline-rich proteins in plants, which support multifaceted cell wall functions beyond simple stabilization, compared to microbial contexts where free hydroxyproline often serves metabolic or regulatory roles. Enzymatic requirements remain mechanistically conserved across kingdoms, but plants feature unique isoform expansions, such as the 13 P4Hs in Arabidopsis, tailored to substrate variability in HRGPs. The ancient evolutionary origin of P4H enzymes, evidenced by functional homologs in prokaryotes like Pseudomonas that predate metazoan collagen systems, underscores their role in early oxygen-sensing mechanisms predating animal-specific adaptations.^[27]

Biological Functions

Role in Collagen and Protein Stability

Hydroxyproline, primarily in its trans-4-hydroxy-L-proline (4R-Hyp) form, plays a central role in the structural integrity of collagen, the most abundant protein in animals, by stabilizing its characteristic triple-helical conformation. Collagen molecules consist of three polypeptide chains arranged in a repeating Gly-X-Y sequence, where hydroxyproline predominantly occupies the Y position, comprising approximately 100 residues per 1000 amino acids in type I collagen, the predominant subtype in skin, bone, and tendons.^[28] This high imino acid content, including hydroxyproline, restricts backbone flexibility and promotes the tight packing required for the triple helix.^[29] The stereospecific incorporation of trans-4-Hyp in the Y position is essential for pre-organizing the proline ring conformation through an inductive electron-withdrawing effect from the hydroxyl group, which favors a Cγ-exo (up) puckering.^[30] This puckering aligns the pyrrolidine ring optimally for the trans peptide bond and the polyproline II helix geometry, enhancing chain rigidity and interchain interactions without relying on direct stereoelectronic effects alone.^[29] In contrast, the cis-4-hydroxyproline (4S-Hyp) isomer induces a Cγ-endo (down) pucker that disrupts helical formation, underscoring the stereochemical precision required for stability across collagen subtypes such as types I, II, and III.^[30] The hydroxyl group of hydroxyproline facilitates stabilization primarily through water-mediated hydrogen bonding networks that bridge adjacent chains in the triple helix, although direct interchain hydrogen bonds also contribute.^[29] This modification increases the melting temperature (T_m) of the collagen triple helix by approximately 15°C compared to unhydroxylated analogs, as observed in type I collagen models, ensuring structural integrity at physiological temperatures.^[31] Furthermore, variations in hydroxyproline content across tissues correlate with tensile strength; for instance, higher hydroxyproline levels in fibrillar collagens enhance mechanical properties, with type I collagen exhibiting superior strength due to its ~10% hydroxyproline composition.^[32] While hydroxyproline is most prominent in collagens, it appears in smaller amounts in non-collagenous proteins such as elastin, where it supports elastic fiber stability, and the complement component C1q, contributing to its collagen-like domain.^[33] However, these roles are minor compared to its dominant function in collagen subtypes, where hydroxyproline content directly influences overall protein stability and tissue biomechanics.^[32]

Metabolic and Signaling Roles

Hydroxyproline serves as a key precursor for glycine synthesis, particularly in neonatal tissues where demand for glycine is high for growth and development. In neonatal pigs, the liver and kidney efficiently convert 4-hydroxyproline to glycine through a dedicated pathway involving enzymes such as hydroxyproline oxidase and Δ¹-pyrroline-5-carboxylate reductase, with renal contributions being prominent due to the organ's role in amino acid metabolism.^[34] This pathway represents a major source of neonatal glycine, highlighting hydroxyproline's metabolic importance beyond its structural integration into collagen proteins.^[35] In energy metabolism and redox signaling, hydroxyproline participates in maintaining cellular homeostasis by influencing mitochondrial function and reactive oxygen species (ROS) balance. Oxidation of hydroxyproline by hydroxyproline oxidase generates ROS and intermediates like glyoxylate and pyruvate, which feed into the tricarboxylic acid cycle, thereby supporting energy production and antioxidative defenses in various cell types.^[36] Furthermore, hydroxyproline modulates redox signaling pathways, including those involving Nrf2 activation, to mitigate oxidative stress and promote cell survival under physiological and pathological conditions.^[37] Hydroxyproline exerts significant influence on cellular signaling, notably in regulating hypoxia-inducible factor-1α (HIF-1α) activity during hypoxic conditions. While prolyl hydroxylation of HIF-1α typically targets it for degradation under normoxia, accumulated hydroxyproline under hypoxia—derived from collagen turnover—upregulates HIF-1α transcriptional activity, enhancing adaptive responses such as glycolysis and angiogenesis.^[3] In cancer contexts, hydroxyproline metabolism amplifies interferon-γ (IFN-γ)-induced programmed death-ligand 1 (PD-L1) expression on myeloid and epithelial cells, thereby potentiating immune evasion mechanisms; this effect stems from hydroxyproline's inhibition of autophagic flux, which disrupts a negative feedback loop on PD-L1.^[38] Emerging roles of hydroxyproline extend to modulation of angiogenesis and apoptosis. In hypoxic microenvironments, such as those in hepatocellular carcinoma, hydroxyproline accumulation from upregulated proline metabolism promotes tumor angiogenesis by stabilizing HIF-1α and enhancing vascular endothelial growth factor signaling, thereby supporting neoplastic progression.^[39] Regarding apoptosis, hydroxyproline oxidase-mediated catabolism of hydroxyproline generates ROS that activate p53-dependent apoptotic cascades, providing a regulatory checkpoint for cell death in response to stress.^[40] Additionally, exogenous hydroxyproline supplementation inhibits autophagy by blocking lysosomal fusion, which has implications for cellular resilience in inflammatory and neoplastic settings.^[38] In non-mammalian systems, dietary hydroxyproline supplementation has demonstrated benefits in aquaculture, particularly for enhancing growth and tissue quality in fish. Studies on triploid crucian carp showed that 0.4-0.8% hydroxyproline in diets improved weight gain by 15-20%, feed efficiency, and muscle collagen content, attributing these effects to boosted proline-hydroxyproline cycling and reduced oxidative damage.^[41]

Catabolism and Metabolism

Degradation Pathways

The degradation of hydroxyproline begins with the breakdown of collagen, its primary source in vertebrates. During tissue remodeling and extracellular matrix turnover, matrix metalloproteinases (MMPs) cleave collagen into smaller peptides containing hydroxyproline residues. These peptides are further processed by prolidase (PEPD), which hydrolyzes the terminal dipeptides to release free trans-4-hydroxy-L-proline (Hyp).^[42] Once freed, Hyp undergoes catabolism primarily in the mitochondria of hepatic and renal cells. The initial step involves oxidation by proline dehydrogenase 2 (PRODH2, also known as hydroxyproline dehydrogenase), converting Hyp to Δ¹-pyrroline-3-hydroxy-5-carboxylate (P3H5C). This intermediate spontaneously hydrolyzes to 4-hydroxyglutamate semialdehyde, which is then oxidized through subsequent enzymatic steps, including conversion to 4-hydroxy-2-oxoglutarate, and ultimately cleavage into glyoxylate and pyruvate by 4-hydroxy-2-oxoglutarate aldolase (HOGA1). The pathway proceeds mitochondrially in vertebrates, with glyoxylate further transaminated to glycine using alanine as the amino donor via alanine-glyoxylate aminotransferase (AGT).^[42]^[43] The overall catabolic equation for Hyp degradation can be summarized as:

\text{Hyp} + \text{alanine} \rightarrow \text{glyoxylate} + \text{pyruvate} + \text{other intermediates leading to glycine}

This process yields equimolar amounts of glyoxylate and pyruvate from the carbon skeleton of Hyp, with net production of glycine for amino acid recycling. Hepatic catabolism predominates in mammals, accounting for the majority of flux, while renal metabolism handles a smaller portion, particularly in response to dietary or turnover-derived Hyp.^[42]^[44]

Associated Enzymes and Regulation

The catabolism of hydroxyproline in vertebrates is initiated by the mitochondrial enzyme hydroxyproline oxidase, also known as proline dehydrogenase 2 (PRODH2) or hydroxyproline dehydrogenase (HYPDH), which catalyzes the oxidation of trans-4-L-hydroxyproline to Δ¹-pyrroline-3-hydroxy-5-carboxylate using FAD as a cofactor and transferring electrons to ubiquinone-10.^[45] This enzyme is encoded by the PRODH2 gene on human chromosome 19q13.12 and exhibits specificity for trans-4-L-hydroxyproline.^[46] The Δ¹-pyrroline-3-hydroxy-5-carboxylate intermediate spontaneously hydrolyzes to 4-hydroxyglutamate semialdehyde, which is then oxidized by the NAD⁺-dependent mitochondrial enzyme aldehyde dehydrogenase 4 family member A1 (ALDH4A1) to 4-hydroxyglutamate.^[47] These enzymes are evolutionarily conserved across vertebrates, with PRODH2 and ALDH4A1 orthologs present in mammals, birds, reptiles, amphibians, and fish, reflecting their essential role in degrading collagen-derived hydroxyproline to prevent metabolic accumulation.^[48] Genetically, PRODH2 and ALDH4A1 do not form tight clusters but are part of broader amino acid metabolism gene families, with PRODH2 showing high sequence similarity to PRODH1 (proline oxidase) across species. Tissue expression of PRODH2 is prominent in the liver and kidney, where cytoplasmic and mitochondrial localization supports hepatic processing of dietary and endogenous hydroxyproline, while lower levels occur in brain and skeletal muscle.^[49] ALDH4A1 follows a similar pattern, with elevated expression in liver and kidney to handle downstream intermediates. Regulatory mechanisms include compartmentalization within mitochondria, which sequesters glyoxylate intermediates away from lactate dehydrogenase to minimize spontaneous conversion to oxalate, thereby providing an intrinsic control on oxalate production from hydroxyproline.^[48] Recent therapeutic research (2022–2025) has explored siRNA approaches to mitigate hyperoxaluria by targeting enzymes in the hydroxyproline-to-oxalate pathway, such as lumasiran (targeting hydroxyacid oxidase 1, HAO1) and nedosiran (targeting lactate dehydrogenase A, LDHA), which reduce urinary oxalate levels derived from hydroxyproline catabolism by 50–70% in clinical trials for primary hyperoxaluria types 1 and 2.^[50] These interventions indirectly modulate hydroxyproline metabolism outcomes without directly silencing PRODH2 or ALDH4A1. Variations in catabolism exist between mammals and fish: in mammals, alanine-glyoxylate aminotransferase (involved downstream) localizes to mitochondria in carnivores and peroxisomes in herbivores, adapting to dietary hydroxyproline loads from collagen, whereas fish rely more on efficient hepatic PRODH2 activity for utilizing dietary hydroxyproline from low-protein feeds, supporting growth without specialized compartmental shifts.^[48]^[1]

Physiological and Clinical Significance

Health and Disease Associations

Hydroxyproline deficiency arises primarily from impaired biosynthesis due to vitamin C (ascorbic acid) deficiency, which serves as a cofactor for prolyl 4-hydroxylase (P4H), the enzyme responsible for hydroxylating proline residues in collagen. Without sufficient hydroxylation, collagen triple helices become unstable, leading to defective connective tissue formation and the clinical manifestations of scurvy, including gingival bleeding, poor wound healing, and subcutaneous hemorrhages. This condition was historically recognized in the 18th century among sailors on long voyages lacking fresh fruits and vegetables, with James Lind's 1747 clinical trial demonstrating citrus fruits' efficacy in prevention.^[51]^[52]^[53] Defects in hydroxyproline catabolism are implicated in primary hyperoxaluria type 3 (PH3), an autosomal recessive disorder caused by mutations in the HOGA1 gene encoding 4-hydroxy-2-oxoglutarate aldolase, leading to accumulation of glyoxylate and excessive oxalate production. This results in recurrent kidney stones and potential nephrocalcinosis, often presenting in infancy or early childhood, though some cases resolve spontaneously. Hydroxyproline metabolism contributes significantly to endogenous oxalate synthesis in PH3, distinguishing it from other hyperoxaluria types.^[54]^[55]^[56] Certain variants of Ehlers-Danlos syndrome (EDS) involve hydroxyproline under-hydroxylation due to deficiencies in P4H activity. For instance, mutations in the P4HA1 gene, which encodes the alpha subunit of P4H, cause a rare congenital disorder of connective tissue characterized by muscle hypotonia, joint hypermobility, scoliosis, and ocular fragility, overlapping with classical EDS features and resulting from unstable collagen structures. Similarly, the spondylocheirodysplastic form of EDS exhibits under-hydroxylated proline residues in collagens despite normal enzyme levels, contributing to short stature, radioulnar synostosis, and skin laxity.^[57]^[58] Elevated urinary hydroxyproline levels serve as an indicator of increased bone resorption in osteoporosis, reflecting accelerated collagen breakdown from bone matrix degradation. In postmenopausal women, higher urinary hydroxyproline correlates with greater bone turnover and fracture risk, though it is less specific than modern markers like pyridinoline crosslinks due to contributions from non-skeletal sources.^[59]^[60] Mutations in P4H genes, particularly P4HA1 and P4HA2, underlie severe collagenopathies by disrupting hydroxyproline formation essential for collagen stability and secretion. These genetic defects lead to phenotypes including myopathy, tendon rupture, and skeletal dysplasia, as seen in recessive disorders with reduced P4H activity causing under-hydroxylated collagen and connective tissue fragility.^[57]^[61]

Biomarkers, Supplementation, and Recent Research

Hydroxyproline serves as a biomarker for collagen turnover, with elevated levels in serum and urine indicating increased bone resorption and remodeling. In conditions like Paget's disease of bone, urinary excretion of peptide-bound hydroxyproline is a widely used marker of heightened bone turnover, often measured alongside serum alkaline phosphatase to monitor disease activity and treatment response.^[62] This marker offers specificity for collagen degradation over total collagen assessments, as hydroxyproline primarily reflects the breakdown of mature collagen fibrils, providing insights into pathological bone dynamics without interference from inflammation in certain contexts.^[63] Supplementation with hydroxyproline has shown benefits in supporting neonatal growth through its conversion to glycine, an essential amino acid for protein synthesis and development in milk-fed infants and piglets. A 2018 review highlighted that hydroxyproline, abundant in milk, undergoes renal metabolism to glycine, aiding neonatal survival and growth when endogenous synthesis is insufficient.^[21] In aquaculture, dietary hydroxyproline supplementation at 0.5-1% in low-fish-meal feeds enhances muscle growth and quality in species like triploid crucian carp, improving weight gain, collagen content, and textural properties such as hardness and chewiness.^[41] Additionally, hydroxyproline as a proline precursor may promote wound healing by boosting collagen synthesis in healing tissues, with targeted amino acid supplementation increasing local hydroxyproline concentrations to accelerate recovery in surgical and diabetic wounds.^[64] Recent research has uncovered hydroxyproline's role in modulating immune responses relevant to cancer immunotherapy, particularly through its enhancement of interferon-gamma-induced PD-L1 expression on tumor and immune cells. A 2023 study demonstrated that hydroxyproline metabolism inhibits autophagic flux, thereby upregulating PD-L1 to promote immune evasion, suggesting potential therapeutic targets for combining hydroxyproline pathway inhibitors with PD-1/PD-L1 blockers.^[38] In hepatocellular carcinoma (HCC), metabolic profiling revealed that hypoxia drives hydroxyproline accumulation via proline metabolism, supporting tumor cell survival and progression under low-oxygen conditions, as shown in a 2018 analysis linking hydroxyproline to HIF-1α stabilization.^[39] For primary hyperoxaluria, a 2025 study explored siRNA-mediated silencing of hydroxyproline oxidase (OH-POX/PRODH2), reducing enzyme activity and oxalate production from hydroxyproline catabolism, offering a novel strategy to mitigate renal damage in this disorder.^[37] Emerging post-2020 studies address gaps in hydroxyproline's non-collagen functions, emphasizing its involvement in redox signaling and energy metabolism beyond structural roles. Hydroxyproline participates in proline cycling to regulate mitochondrial redox balance and ATP production, linking amino acid catabolism to cellular stress responses in cancer and fibrosis.^[65] These findings highlight hydroxyproline's regulatory axis with hypoxia-inducible factors and autophagy, influencing energy homeostasis and tumor microenvironment dynamics in ways that extend to therapeutic reprogramming.^[3]

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Asymmetric Syntheses of All Stereoisomers of 3-Hydroxyproline
Aug 10, 2025 · Several synthetic strategies have been developed to obtain stereoisomers of 3-hydroxyproline, including asymmetric [12, 13] and chemoenzimatic ...
[15]
Hydroxyproline
Constituent of collagen, q.v. Isoln from gelatin: E. Fischer, Ber. 35, 2660 (1902). Synthesis: H. Leuchs, Ber. 38, 1937 (1905); and structure determn: H ...Missing: isolated | Show results with:isolated
[16]
https://doi.org/10.1073/pnas.53.2.335
[17]
https://doi.org/10.1073/pnas.53.3.661
[18]
Prolyl 4 Hydroxylase: A Critical Target in the Pathophysiology of ...
These C-P4Hs have a central role in the biosynthesis of collagens as 4-hydroxyproline residues are essential for the formation of the collagen triple helix. 1.
[19]
Collagen prolyl 4-hydroxylase 1 is essential for HIF-1α stabilization ...
Oct 26, 2018 · Collagen prolyl 4-hydroxylase (P4H) is an α2β2 tetrameric α-ketoglutarate (α-KG)-dependent dioxygenase that catalyzes 4-hydroxylation of proline ...
[20]
Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells
May 12, 2000 · Our results indicate that PHalpha(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.<|control11|><|separator|>
[21]
Metabolism, Nutrition, and Redox Signaling of Hydroxyproline
The content of hydroxyproline (Hyp) residue in collagen is 10.84% (g/g). The rates of degradation, net synthesis, and total synthesis of collagen in the body ...
[22]
Bioinformatic Identification of Plant Hydroxyproline-Rich Glycoproteins
Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development.
[23]
Biosynthesis of cell wall protein: Sequential hydroxylation of proline ...
The biosynthesis of the hydroxyproline-rich cell wall protein involves several discrete cytoplasmic steps: the assembly of the polypeptide chain; ...
[24]
An Arabidopsis Prolyl 4 Hydroxylase Is Involved in the Low Oxygen ...
Mar 14, 2021 · These results indicate active involvement of proline hydroxylation, a post-translational modification, to low oxygen response in Arabidopsis.
[25]
Biosynthesis of trans-4-hydroxyproline by recombinant strains of ...
May 19, 2014 · In animal tissue, 4-hydroxyproline is catalyzed by prolyl 4-hydroxylase, which takes peptidyl proline as substrate rather than free proline [6].
[26]
Cell wall regeneration in Chlamydomonas: accumulation of mRNAs ...
The unicellular alga Chlamydomonas reinhardtii is surrounded by a cell wall composed entirely of hydroxyproline-rich glycoproteins (HRGPs).
[27]
Human oxygen sensing may have origins in prokaryotic elongation ...
The finding that a PHD-related prolyl-hydroxylase in Pseudomonas spp. regulates pyocyanin biosynthesis supports prokaryotic origins for the eukaryotic prolyl- ...
[28]
Identification of a highly stable bioactive 3-hydroxyproline ... - NIH
Jun 3, 2022 · Our data showed that Gly-3Hyp-4Hyp has chemotactic activity on skin fibroblasts. Hyp is suggested to be an essential constituent for collagen- ...
[29]
COLLAGEN STRUCTURE AND STABILITY - PMC - NIH
The amino acids in the Xaa and Yaa positions of collagen are often (2S)-proline (Pro, 28%) and (2S,4R)-4-hydroxyproline (Hyp, 38%), respectively. ProHypGly is ...
[30]
Collagen Structure and Stability - Annual Reviews
Apr 3, 2009 · (b) A gauche effect, elicited by an electron- withdrawing group (EWG) in the 4R position, stabilizes the Cγ-exo ring pucker. (c) An n→π∗ ...
[31]
Differential Tensile Strength and Collagen Composition in ... - NIH
Collagen content, as estimated by hydroxyproline, as a function of tensile strength for each TAV (A) and BAV (B) specimen tested. Note a moderate ...
[32]
Hydroxyproline Reference Range
May 5, 2022 · Small amounts of hydroxyproline are also found in the following: Elastin. Acetylcholinesterase. The C1q component of the complement system.
[33]
Synthesis of glycine from 4-hydroxyproline in tissues of neonatal ...
This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues ...
[34]
Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs
Glycine was synthesized from 0.1 to 5 mM 4-hydroxyproline in the liver and kidney from 0- to 21-day-old piglets, as well as the pancreas, small intestine, and ...
[35]
Metabolism, Nutrition, and Redox Signaling of Hydroxyproline
Feb 1, 2019 · Understanding the mechanisms whereby hydroxyproline participates in metabolism and cell signaling can improve the nutrition and health of ...Missing: energy | Show results with:energy
[36]
The new insight into the role of hydroxyproline in metabolism of ...
May 15, 2025 · Hyp is involved in energy and redox homeostasis, DNA synthesis, activation of some transcription factors and epigenetic regulation. These ...
[37]
The new insight into the role of hydroxyproline in metabolism of ...
May 16, 2025 · Of great importance is the role of Hyp in upregulation of transcriptional activity of hypoxia-inducible factor 1α (HIF-1α), inducing ...
[38]
Hydroxyproline metabolism enhances IFN-γ-induced PD-L1 ...
Sep 21, 2023 · We have determined that hydroxyproline (Hyp) significantly and directly enhances adaptive (ie, IFN-γ-induced) PD-L1 expression in multiple relevant myeloid and ...<|separator|>
[39]
Global Metabolic Profiling Identifies a Pivotal Role of Proline and ...
Our results demonstrate that hypoxic microenvironment activates proline metabolism, resulting in accumulation of hydroxyproline that promotes HCC tumor ...<|separator|>
[40]
A Novel Function for Hydroxyproline Oxidase in Apoptosis through ...
We hypothesize that OH-POX plays a role analogous to POX in growth regulation, ROS generation, and activation of the apoptotic cascade.
[41]
Dietary Supplementation With Hydroxyproline Enhances Growth ...
May 31, 2022 · Dietary supplementation of Hyp can enhance fish growth performance, collagen production, muscle textural characteristics and muscle growth of ITCC.
[42]
https://doi.org/10.3390/ijms23021005
[43]
https://doi.org/10.1007/s00726-021-03056-x
[44]
https://doi.org/10.1016/S0174-173X(87)80001-8
[45]
Proline dehydrogenase 2 (PRODH2) is a hydroxyproline ... - NIH
This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C- ...
[46]
PRODH2 Gene - GeneCards | HYPDH Protein
PRODH2 encodes a protein that catalyzes the first step in the catabolism of trans-4-hydroxy-L-proline, an amino acid derivative.
[47]
Disorders of Proline and Hydroxyproline Metabolism - OMMBID
The hydroxyproline degradation pathway resembles that of proline. Δ1-Pyrroline-3-hydroxy-5-carboxylate, the oxidation product of hydroxyproline, is ...Missing: equation | Show results with:equation
[48]
Catabolism of Hydroxyproline in Vertebrates: Physiology, Evolution ...
Jan 17, 2022 · Hydroxylation at position 3 is rare and is mainly found in type I and type IV collagens, with an occurrence of 2 to 10 per 1000 amino acid ...
[49]
Tissue expression of PRODH2 - Summary - The Human Protein Atlas
Summary of PRODH2 (HSPOX1) expression in human tissue. Cytoplasmic expression in kidney and liver.Missing: hydroxyproline | Show results with:hydroxyproline<|separator|>
[50]
Nedosiran, a Candidate siRNA Drug for the Treatment of ... - NIH
Primary hyperoxaluria (PH) is a group of rare genetic disorders associated with chronic kidney diseases and renal failure caused by kidney stones from ...
[51]
Scurvy: Rediscovering a Forgotten Disease - PMC - NIH
May 26, 2023 · Scurvy is a nutritional deficiency caused by low vitamin C levels that has been described since ancient times.
[52]
Vitamin C Deficiency - StatPearls - NCBI Bookshelf
Nov 12, 2023 · Scurvy is a clinical syndrome resulting from vitamin C deficiency. Vitamin C is essential for the growth and repair of skin, cartilage, bone ...
[53]
Ascorbate requirement for hydroxylation and secretion of procollagen
Vitamin C deficiency is associated with defective connective tissue, particularly in wound healing. Ascorbate is required for hydroxylation of proline ...
[54]
Hydroxyproline metabolism in a mouse model of Primary ... - NIH
Primary Hyperoxaluria Type 3 is a recently discovered form of this autosomal recessive disease that results from mutations in the gene coding for 4-hydroxy-2- ...
[55]
4-Hydroxyglutamate Is a Biomarker for Primary Hyperoxaluria Type 3
Primary hyperoxaluria type 3 (PH3) is a recently identified inborn error of 4-hydroxyproline metabolism causing kidney stone disease.
[56]
Hydroxyproline Metabolism and Oxalate Synthesis in Primary ... - NIH
Hydroxyproline (Hyp) metabolism results in the formation of oxalate and glycolate. However, the relative contribution of Hyp metabolism to endogenous oxalate ...
[57]
P4HA1 mutations cause a unique congenital disorder of connective ...
Mutations affecting C-P4Hs should be considered in patients presenting with connective tissue/myopathy overlap disorders with features of Ehlers-Danlos ...
[58]
Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome
Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a ...
[59]
Effect of Antiresorptive Therapy on Urinary Hydroxyproline in ... - NIH
Typically, the urinary markers of bone resorption include hydroxyproline, which serve as rapid predictors of changes in collagen metabolism [5]. Hydroxyproline ...
[60]
Biochemical Markers of Bone Turnover Part I - NIH
Urinary OHP is usually considered to reflect bone resorption. However, it should be noted that significant amounts of urinary OHP are derived from the ...
[61]
Role of prolyl hydroxylation in the molecular interactions of collagens
Proline hydroxylation may regulate the flexibility of collagen molecules and make functional sites available for interacting proteins and receptors. Collagens ...<|control11|><|separator|>
[62]
Biochemical markers of bone turnover in Paget's disease - PubMed
The two most commonly used biochemical markers of bone turnover are the serum alkaline phosphatase and the urinary excretion of peptide-bound hydroxyproline.Missing: biomarker | Show results with:biomarker
[63]
Urinary Hydroxyproline as an Inflammation-Independent Biomarker ...
Jun 6, 2024 · This study identifies urinary hydroxyproline, a marker of collagen turnover elevated in experimental colitis, as independent of conventional biomarkers.
[64]
Proline Precursors and Collagen Synthesis: Biochemical ... - NIH
Both proline and hydroxyproline are vital for collagen biosynthesis, structure, and strength. Their cyclic structure constrains the rotation of the polypeptide ...
[65]
the proline cycle/proline-collagen regulatory axis | Amino Acids
Nov 26, 2021 · Thus, the proline metabolic axis may provide a regulatory connection to link energy metabolism, redox regulation, collagen synthesis, the ...