Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies.

Screening for pharmaceutically viable stability from measurements of thermally induced protein unfolding and short-term accelerated stress underpins much molecule design, selection, and formulation in the pharmaceutical biotechnology industry. However, the interrelationships among intrinsic protein conformational stability, thermal denaturation, and pharmaceutical stability are complex. There are few publications in which predictions from thermal unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight developable therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under common reference buffer conditions and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5, 25, and 40 °C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. Our data suggest that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition may be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.

Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress.

Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, alpha-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes.

Effects of solution conditions and surface chemistry on the adsorption of three recombinant botulinum neurotoxin antigens to aluminum salt adjuvants.

Botulinum neurotoxin (BoNT) is a biological warfare threat. Protein antigens have been developed against the seven major BoNT serotypes for the development of a recombinant protein vaccine. This study is an evaluation of adsorption profiles for three of the recombinant protein antigens to aluminum salt adjuvants in the development of a trivalent vaccine against BoNT. Adsorption profiles were obtained over a range of protein concentrations. The results document that charge-charge interactions dominate the adsorption of antigen to adjuvant. Optimal conditions for adsorption were determined. However, potency studies and solution stability studies indicated the necessity of using aluminum hydroxide adjuvant at low pH. To improve the adsorption profiles to AlOH adjuvant, phosphate ions were introduced into the adsorption buffers. The resulting change in the adjuvant chemistry led to an improvement of adsorption of the BoNT antigens to aluminum hydroxide adjuvant while maintaining potency. Competitive adsorption profiles were also determined, and showed changes in maximum adsorption from mixed solutions compared to adsorption from individual protein solutions. The adsorption profiles for each protein varied due to differences in adsorption mechanism and affinity for the adjuvant surface. These results emphasize the importance of evaluating competitive adsorption in the development of multivalent vaccine products.

Assessing the Impact of Charge Variants on Stability and Viscosity of a High Concentration Antibody Formulation.

Characterizing molecular charge variants or isoforms is essential for understanding safety, potency, and bioavailability of antibody therapeutics. However, there is little information on how they influence stability and viscosity-properties governing immunogenicity and delivery. To bridge this gap, we studied antibody stability as a function of charge variant content generated via bioreactor process. We were able to systematically vary acidic variant levels as a function of bioreactor harvest time. Importantly, we do not observe any impact on aggregation behavior of a formulated antibody at high protein concentration as a function of acidic variant level. Furthermore, we confirm that acidic variants enriched using fractionation do not influence viscosity, colloidal or conformational stability. Interestingly, variants with the most acidic isoelectric points contribute disproportionately to formulation color. We discuss our findings in context of antibody manufacturing processes that may yield increased charge variant content.

BSA degradation under acidic conditions: a model for protein instability during release from PLGA delivery systems.

Acidification of the internal poly(lactide-co-glycolide) (PLGA) microenvironment is considered one of the major protein stresses during controlled release from such delivery systems. A model protein, bovine serum albumin (BSA), was incubated at 37 degrees C for 28 days to simulate the environment within the aqueous pores of PLGA during the release phase and to determine how acidic microclimate conditions affect BSA stability. Size-exclusion high performance liquid chromatography (SE-HPLC), SDS-PAGE, and infrared spectroscopy were used to monitor BSA degradation. BSA was most stable at pH 7, but rapidly degraded via aggregation and hydrolysis at pH 2. These simulated degradation products were nearly identical to that of unreleased BSA found entrapped within PLGA 50/50 millicylinders. At pH 2, changes in BSA conformation detected by various spectroscopic techniques were consistent with acid denaturation of the protein. By contrast, at pH 5 and above, damage to BSA was insufficient to explain the instability of the protein in the polymer. Thus, these data confirm the hypothesis that acid-induced unfolding is the basis of BSA aggregation in PLGA and the acidic microclimate within PLGA is indeed a dominant stress for encapsulated BSA. To increase the stability of proteins within PLGA systems, formulations must protect against potentially extreme acidification such that native structure is maintained.

Human aldehyde dehydrogenase 3A1 (ALDH3A1): biochemical characterization and immunohistochemical localization in the cornea.

ALDH3A1 (aldehyde dehydrogenase 3A1) is expressed at high concentrations in the mammalian cornea and it is believed that it protects this vital tissue and the rest of the eye against UV-light-induced damage. The precise biological function(s) and cellular distribution of ALDH3A1 in the corneal tissue remain to be elucidated. Among the hypotheses proposed for ALDH3A1 function in cornea is detoxification of aldehydes formed during UV-induced lipid peroxidation. To investigate in detail the biochemical properties and distribution of this protein in the human cornea, we expressed human ALDH3A1 in Sf9 insect cells using a baculovirus vector and raised monoclonal antibodies against ALDH3A1. Recombinant ALDH3A1 protein was purified to homogeneity with a single-step affinity chromatography method using 5'-AMP-Sepharose 4B. Human ALDH3A1 demonstrated high substrate specificity for medium-chain (6 carbons and more) saturated and unsaturated aldehydes, including 4-hydroxy-2-nonenal, which are generated by the peroxidation of cellular lipids. Short-chain aliphatic aldehydes, such as acetaldehyde, propionaldehyde and malondialdehyde, were found to be very poor substrates for human ALDH3A1. In addition, ALDH3A1 metabolized glyceraldehyde poorly and did not metabolize glucose 6-phosphate, 6-phosphoglucono-delta-lactone and 6-phosphogluconate at all, suggesting that this enzyme is not involved in either glycolysis or the pentose phosphate pathway. Immunohistochemistry in human corneas, using the monoclonal antibodies described herein, revealed ALDH3A1 expression in epithelial cells and stromal keratocytes, but not in endothelial cells. Overall, these cumulative findings support the metabolic function of ALDH3A1 as a part of a corneal cellular defence mechanism against oxidative damage caused by aldehydic products of lipid peroxidation. Both recombinant human ALDH3A1 and the highly specific monoclonal antibodies described in the present paper may prove to be useful in probing biological functions of this protein in ocular tissue.

Molecular cloning and baculovirus expression of the rabbit corneal aldehyde dehydrogenase (ALDH1A1) cDNA.

Most mammalian species express high concentrations of ALDH3A1 in corneal epithelium with the exception of the rabbit, which expresses high amounts of ALDH1A1 rather than ALDH3A1. Several hypotheses that involve catalytic and/or structural functions have been postulated regarding the role of these corneal ALDHs. The aim of the present study was to characterize the biochemical properties of the rabbit ALDH1A1. We have cloned and sequenced the rabbit ALDH1A1 cDNA, which is 2,073 bp in length (excluding the poly(A+) tail), and has 5' and 3' nontranslated regions of 46 and 536 bp, respectively. This ALDH1A1 cDNA encodes a protein of 496 amino acids (Mr = 54,340) that is: 86-91% identical to mammalian ALDH1A1 proteins, 83-85% identical to phenobarbital-inducible mouse and rat ALDH1A7 proteins, 84% identical to elephant shrew ALDH1A8 proteins (eta-crystallins), 69-73% identical to vertebrate ALDH1A2 and ALDH1A3 proteins, 65% identical to scallop ALDH1A9 protein (omega-crystallin), and 55-57% to cephalopod ALDH1C1 and ALDH1C2 (omega-crystallins). Recombinant rabbit ALDH1A1 protein was expressed using the baculovirus system and purified to homogeneity with affinity chromatography. We found that rabbit ALDH1A1 is catalytically active and efficiently oxidizes hexanal (Km = 3.5 microM), 4-hydroxynonenal (Km = 2.1 microM) and malondialdehyde (Km = 14.0 microM), which are among the major products of lipid peroxidation. Similar kinetic constants were observed with the human recombinant ALDH1A1 protein, which was expressed and purified using similar experimental conditions. These data suggest that ALDH1A1 may contribute to corneal cellular defense against oxidative damage by metabolizing toxic aldehydes produced during UV-induced lipid peroxidation.

Ultraviolet radiation decreases expression and induces aggregation of corneal ALDH3A1.

Substantial reduction in corneal ALDH3A1 enzymatic activity associated with eye pathology was previously reported in C57BL/6J mice subjected to ultraviolet radiation (UVR). The aim of this study was to examine whether UVR diminishes corneal ALDH3A1 expression through modifications at the transcriptional, translational, or post-translational level. Adult C57BL/6J mice were subjected to UVR exposure (302 nm peak wavelength) for various periods of time, and corneal ALDH3A1 mRNA and protein levels were monitored by Northern and Western blot analysis, respectively. In addition, ALDH3A1 enzymatic activity was determined as a measure of post-translational modification. Mice exposed to 0.2 J/cm(2) UVB radiation demonstrated an extensive decrease, approximately 80%, in mRNA and protein levels, as well as enzymatic activity of corneal ALDH3A1. Significant reductions in corneal ALDH3A1 enzymatic activity were detected in mice 96 h after exposure to 0.05 and 0.1 J/cm(2) UVB radiation; no significant changes were observed in mRNA and protein levels. These data suggest that UVB down-regulates corneal ALDH3A1 expression at the transcriptional and/or post-translational level depending on the dose of UVB. Reduction in gene transcription requires UVB doses greater than or equal to 0.2 J/cm(2). In vitro experiments with human corneal epithelial cell lines stably transfected with human ALDH3A1 cDNA, and with purified recombinant human ALDH3A1 protein, indicated that ALDH3A1 undergoes post-translational modifications after UVR exposure. These modifications result in both covalent and non-covalent aggregation of the protein with no detectable precipitation. Such conformational changes may be associated with the function of ALDH3A1 as a chaperone-like molecule in the cornea.

Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light.

As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys.

Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.

Vaccines utilizing recombinant protein antigens typically require an adjuvant to enhance immune response in the recipients. However, the consequences of antigen binding to adjuvant on both the short- and long-term stability of the protein remain poorly defined. In our companion paper (Vessely et al., in press, J Pharm Sci), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(H(c)), rBoNTB(H(c)), and rBoNTE(H(c)). In the current study, we evaluated the effect of binding to adjuvant (Alhydrogel, aluminum hydroxide) on chemical stability of these antigens during long-term storage in aqueous suspension. We developed methods that employ LysC peptide mapping in conjunction with MALDI-TOF mass spectrometry. Peptide maps were developed for the proteins for a vaccine formulation of rBoNTE(H(c)) as well as a trivalent rBoNT(H(c)) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a postdigestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(H(c)) protein antigens. We found that all three of the rBoNT(H(c)) proteins were susceptible to degradation via both oxidation and deamidation. In many cases, such reactions occurred earlier with the adjuvant-bound protein formulations when compared to the proteins in control samples that were not bound to adjuvant. Additionally, some chemical modifications were found in the adjuvant-bound protein formulations but were not detected in the unbound solution controls. Our studies indicate that binding to aluminum-based adjuvants can impact the chemical stability and/or the chemical degradation pathways of protein during long-term storage in aqueous suspension. Furthermore, the methods we developed should be widely useful for assessing chemical stability of adjuvant-bound recombinant protein antigens.

Stability of a trivalent recombinant protein vaccine formulation against botulinum neurotoxin during storage in aqueous solution.

The adsorption of recombinant botulinum neurotoxin (BoNT) protein-derived vaccine antigens to aluminum salt adjuvants has been previously studied for the development of a trivalent vaccine against the neurotoxins (Vessely et al., in press, J Pharm Sci). The current paper describes an investigation of the stability of recombinant BoNT antigens adsorbed to aluminum salt adjuvants during storage in aqueous solution. Both chemical and physical changes occurred during storage. Phosphate groups in the buffer exchanged with hydroxyl groups on the adjuvant surface. The resulting changes in solution pH and adjuvant surface chemistry promoted more favorable electrostatic interaction between the BoNT proteins and the surface, possibly increasing the affinity of the proteins for the surface during storage. Fluorescence and UV spectroscopy suggested changes to protein structure during storage, whereas differential scanning calorimetry showed changes to thermal processes related to protein conformation and/or surface adsorption. The consequence of the chemical and physical changes to the proteins was a decrease in the ability to desorb protein from the adjuvant surface during storage. Overall, the results of this study emphasize the utility of a thorough characterization of the interactions between protein antigens and aluminum salt adjuvants.

The role of corneal crystallins in the cellular defense mechanisms against oxidative stress.

The refracton hypothesis describes the lens and cornea together as a functional unit that provides the proper ocular transparent and refractive properties for the basis of normal vision. Similarities between the lens and corneal crystallins also suggest that both elements of the refracton may also contribute to the antioxidant defenses of the entire eye. The cornea is the primary physical barrier against environmental assault to the eye and functions as a dominant filter of UV radiation. It is routinely exposed to reactive oxygen species (ROS)-generating UV light and molecular O(2) making it a target vulnerable to UV-induced damage. The cornea is equipped with several defensive mechanisms to counteract the deleterious effects of UV-induced oxidative damage. These comprise both non-enzymatic elements that include proteins and low molecular weight compounds (ferritin, glutathione, NAD(P)H, ascorbate and alpha-tocopherol) as well as various enzymes (catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase, and superoxide dismutase). Several proteins accumulate in the cornea at unusually high concentrations and have been classified as corneal crystallins based on the analogy of these proteins with the abundant taxon-specific lens crystallins. In addition to performing a structural role related to ocular transparency, corneal crystallins may also contribute to the corneal antioxidant systems through a variety of mechanisms including the direct scavenging of free radicals, the production of NAD(P)H, the metabolism and/or detoxification of toxic compounds (i.e. reactive aldehydes), and the direct absorption of UV radiation. In this review, we extend the discussion of the antioxidant defenses of the cornea to include these highly expressed corneal crystallins and address their specific capacities to minimize oxidative damage.

ALDH3A1: a corneal crystallin with diverse functions.

Aldehyde dehydrogenase 3A1 (ALDH3A1) comprises a surprisingly high proportion (5-50% depending on species) of the water-soluble protein of the mammalian cornea, but is present little if at all in the cornea of other species. Mounting experimental evidence demonstrates that this abundant corneal protein plays an important role in the protection of ocular structures against oxidative damage. Corneal ALDH3A1 appears to protect against UV-induced oxidative stress through a variety of biological functions such as the metabolism of toxic aldehydes produced during the peroxidation of cellular lipids, the generation of the antioxidant NADPH, the direct absorption of UV-light, the scavenging of reactive oxygen species (ROS), and the possession of chaperone-like activity. With analogies to the abundant, multifunctional, and taxon-specific lens crystallins, mammalian ALDH3A1 has been considered a corneal crystallin, suggesting that it may contribute to the optical properties of the cornea as well. Recent studies have also revealed a novel role for ALDH3A1 in the regulation of the cell cycle. ALDH3A1-transfected HCE cells have increased population-doubling time, decreased plating efficiency, and reduced DNA synthesis, most likely due to a profound inhibition of cyclins and cyclin-dependent kinases. We have proposed that the ALDH3A1-induced reduction in cell growth may contribute to protection against oxidative stress by extending time for DNA and cell repair. Taken together, the multiple roles of ALDH3A1 against oxidative stress in addition to its contributions to the optical properties of the cornea are consistent with the idea that this specialized protein performs diverse biological functions as do the lens crystallins.

Mechanisms involved in the protection of UV-induced protein inactivation by the corneal crystallin ALDH3A1.

Various lines of evidence have shown that ALDH3A1 (aldehyde dehydrogenase 3A1) plays a critical and multifaceted role in protecting the cornea from UV-induced oxidative stress. ALDH3A1 is a corneal crystallin, which is defined as a protein recruited into the cornea for structural purposes without losing its primary function (i.e. metabolism). Although the primary role of ALDH3A1 in the metabolism of toxic aldehydes has been clearly demonstrated, including the detoxification of aldehydes produced during UV-induced lipid peroxidation, the structural role of ALDH3A1 in the cornea remains elusive. We therefore examined the potential contribution of ALDH3A1 in maintaining the optical integrity of the cornea by suppressing the aggregation and/or inactivation of other proteins through chaperone-like activity and other protective mechanisms. We found that ALDH3A1 underwent a structural transition near physiological temperatures to form a partially unfolded conformation that is suggestive of chaperone activity. Although this structural transition alone did not correlate with any protection, ALDH3A1 substantially reduced the inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal and malondialdehyde when co-incubated with NADP(+), reinforcing the importance of the metabolic function of this corneal enzyme in the detoxification of toxic aldehydes. A large excess of ALDH3A1 also protected glucose-6-phosphate dehydrogenase from inactivation because of direct exposure to UVB light, which suggests that ALDH3A1 may shield other proteins from damaging UV rays. Collectively, these data demonstrate that ALDH3A1 can reduce protein inactivation and/or aggregation not only by detoxification of reactive aldehydes but also by directly absorbing UV energy. This study provides for the first time mechanistic evidence supporting the structural role of the corneal crystallin ALDH3A1 as a UV-absorbing constituent of the cornea.

Multiple and additive functions of ALDH3A1 and ALDH1A1: cataract phenotype and ocular oxidative damage in Aldh3a1(-/-)/Aldh1a1(-/-) knock-out mice.

ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(-/-)/Aldh3a1(-/-) double knock-out and Aldh1a1(-/-) and Aldh3a1(-/-) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1- and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6-9 months of age). One- to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(-/-) and Aldh3a1(-/-)/Aldh1a1(-/-) mice compared with Aldh1a1(-/-) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal- and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.

Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2.

Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway and, to a lesser extent, through microsomal oxidation (CYP2E1) and the catalase-H(2)O(2) system. Acetaldehyde, which is responsible for some of the deleterious effects of ethanol, is further oxidized to acetic acid by aldehyde dehydrogenases (ALDHs), of which mitochondrial ALDH2 is the most efficient. The aim of this study was to evaluate zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding a protein of 516 amino acids (M(r) = 56,562), approximately 75% identical to mammalian ALDH2 proteins. Recombinant zebrafish ALDH2 protein was expressed using the baculovirus expression system and purified to homogeneity by affinity chromatography. We found that zebrafish ALDH2 is catalytically active and efficiently oxidizes acetaldehyde (K(m) = 11.5 microM) and propionaldehyde (K(m) = 6.1 microM). Similar kinetic properties were observed with the recombinant human ALDH2 protein, which was expressed and purified using comparable experimental conditions. Western blot analysis revealed that ALDH2 is highly expressed in the heart, skeletal muscle, and brain with moderate expression in liver, eye, and swim bladder of the zebrafish. These results are the first reported on the cloning, expression, and characterization of a zebrafish ALDH, and indicate that zebrafish is a suitable model for studying ethanol metabolism and, therefore, toxicity.

Role of human aldehyde dehydrogenases in endobiotic and xenobiotic metabolism.

The human genome contains at least 17 genes that are members of the aldehyde dehydrogenase (ALDH) superfamily. These genes encode NAD(P)(+)-dependent enzymes that oxidize a wide range of aldehydes to their corresponding carboxylic acids. Aldehydes are highly reactive molecules that are intermediates or products involved in a broad spectrum of physiologic, biologic, and pharmacologic processes. Aldehydes are generated during retinoic acid biosynthesis and the metabolism of amino acids, lipids, carbohydrates, and drugs. Mutations in several ALDH genes are the molecular basis of inborn errors of metabolism and contribute to environmentally induced diseases.