Step In Vitamin Research Articles

Pathophysiological significance of senescence marker protein‐30

A novel rat liver protein of 30 kDa, SMP30 decreases with aging. This protein is expressed most prominently in the liver and kidneys among the various organs. Its gene is located on the X chromosome. No functional domain was recognized in the entire amino acid sequence. Recently, we found a homology between rat SMP30 and two species of bacterial gluconolactonase (EC 3.1.1.17). The lactonase reaction with L-gulono-gamma-lactone is the penultimate step in vitamin C (L-ascorbic acid) biosynthesis. SMP30-knockout (KO) mice fed a vitamin C-deficient diet displayed symptoms of scurvy. In SMP30-KO mice, hepatocytes were more susceptible to apoptosis induced by TNF-alpha plus actinomycin D than hepatocytes from wild-type mice. Two morphological features considered to be a hallmark of senescence are apparent in SMP30-KO mice. At 12 months of age, SMP30-knockout mice had clearly visible deposits of lipofuscin and senescence-associated beta-galactosidase (SA-beta-GAL) in their renal tubular epithelia. These features are compatible with high electron dense deposits in lysosomes. This observation suggests that the SMP30-knockout mouse is a useful model of ordinal senescence.

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

Backgroundβ-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system.ResultsOur assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.ConclusionsOur data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.

Significance of SMP30 in gerontology

The expression of senescence marker protein‐30 (SMP30) was discovered by proteomic analysis. The fact that this molecule's structure is so highly conserved among numerous species strongly suggests that the age‐dependent decrease of SMP30 may contribute to senescence. Research that targeted the SMP30 gene showed deterioration in several organs accompanied by a shortened life span. We then identified SMP30 as gluconolactonase (GNL). The lactonase reaction with L‐gulono‐γ‐lactone is the penultimate step in vitamin C (L‐ascorbic acid) biosynthesis. Our discovery has opened the door to a new aspect of aging studies.

The terminal step in vitamin C biosynthesis in Trypanosoma cruzi is mediated by a FMN-dependent galactonolactone oxidase.

Humans lack the ability to synthesize vitamin C (ascorbate) due to the absence of gulonolactone oxidase, the last enzyme in the biosynthetic pathway in most other mammals. The corresponding oxidoreductase in trypanosomes therefore represents a target that may be therapeutically exploitable. This is reinforced by our observation that Trypanosoma cruzi, the causative agent of Chagas' disease, lacks the capacity to scavenge ascorbate from its environment and is therefore dependent on biosynthesis to maintain intracellular levels of this vitamin. Here, we show that T. cruzi galactonolactone oxidase (TcGAL) can utilize both L-galactono-gamma-lactone and D-arabinono-gamma-lactone as substrates for synthesis of vitamin C, in reactions that obey Michaelis-Menten kinetics. It is >20-fold more active than the analogous enzyme from the African trypanosome Trypanosoma brucei. FMN is an essential cofactor for enzyme activity and binds to TcGAL non-covalently. In other flavoproteins, a histidine residue located within the N-terminal flavin-binding motif has been shown to be crucial for cofactor binding. Using site-directed mutagenesis, we show that the corresponding residue in TcGAL (Lys-55) is not essential for this interaction. In contrast, we find that histidine and tryptophan residues (His-447 and Trp-448), localized within a C-terminal motif (HWXK) that is a feature of ascorbate-synthesizing enzymes, are necessary for the FMN association. The conserved lysine residue within this motif (Lys-450) is not required for cofactor binding, but its replacement by glycine renders the protein completely inactive.

PHOSPHORUS METABOLISM AND MANAGEMENT IN CHRONIC KIDNEY DISEASE: Expanding Role for Vitamin D in Chronic Kidney Disease: Importance of Blood 25‐OH‐D Levels and Extra‐Renal 1α‐Hydroxylase in the Classical and Nonclassical Actions of 1α,25‐Dihydroxyvitamin D3

AbstractRecent advances in the understanding of vitamin D have revolutionized our view of this old nutritional factor and suggested that it has much wider effects on the body than ever believed before. In addition to its well‐known effects on calcium/phosphate homeostasis, vitamin D, through its hormonal form, 1α,25‐dihydroxyvitamin D3 or calcitriol, is a cell differentiating factor and anti‐proliferative agent with actions on a variety of tissues around the body (e.g., skin, muscle, immune system). By influencing gene expression in multiple tissues, calcitriol influences many physiological processes besides calcium/phosphate homeostasis including muscle and keratinocyte differentiation, insulin secretion, blood pressure regulation, and the immune response. The incidence of various diseases including epithelial cancers, multiple sclerosis, muscle weakness as well as bone‐related disorders has been correlated with vitamin D deficiency/insufficiency and has led to a re‐evaluation of recommended daily intakes both in the normal subject and CKD patient. Critical developments have been the emergence of the value of blood 25‐OH‐D measurement as a tool in predicting vitamin D‐related problems and this has in turn emphasized the importance of the extra‐renal version of the 1α‐hydroxylase, the enzyme responsible for the final step in vitamin D activation. The widespread expression of this extra‐renal enzyme supports the view that it exists to boost intracellular concentrations of calcitriol within some target tissues in order to modulate a unique set of genes specifically in those tissues, a process which is therefore dependent upon circulating 25‐OH‐D. For CKD patients with their tendency to vitamin D substrate insufficiency coupled with their documented loss of the renal 1α‐hydroxylase in late stages, this new information has profound implications. Physicians must start to manage the vitamin D insufficiency by vitamin D supplements throughout stages 1–5 whilst continuing to provide calcitriol replacement therapy, in the form of calcitriol or its analogs, in stages 3–5.

Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana

Vitamin E is an essential nutrient for humans and is obtained primarily from food, especially oil, derived from the seed of plants. Genes encoding the committed steps in vitamin E synthesis in plants (VTE, loci 1-5) have been isolated and used for tocopherol pathway engineering with various degrees of success. As a complement to such approaches we have used quantitative trait loci analysis with two sets of Arabidopsis thaliana recombinant inbred lines and have identified 14 QVE (quantitative vitamin E) loci affecting tocopherol content and composition in seeds. Five QVE intervals contain VTE loci that are likely QVE gene candidates. Nine QVE intervals do not contain VTE loci and therefore identify novel loci affecting seed tocopherol content and composition. Several near-isogenic lines containing introgressions of the accession with increased vitamin E levels were shown to confer significantly elevated tocopherol levels compared with the recurrent parent. Fine-mapping has narrowed QVE7 (a gamma-tocopherol quantitative trait loci) to an 8.5-kb interval encompassing two genes. Understanding the basis of the QVE loci in Arabidopsis promises to provide insight into the regulation and/or metabolism of vitamin E in plants and has clear ramifications for improving the nutritional content of crops through marker-assisted selection and metabolic engineering.

Molecular cloning of the rat β-carotene 15,15′-monooxygenase gene and its regulation by retinoic acid

beta-Carotene exhibits biological activity as provitamin A. Key step in vitamin A formation is the cleavage of beta-carotene to retinal by an enzyme designated as beta-carotene 15,15'-monooxygenase (BCM). Recently, it is reported that expression of BCM gene in the intestine is under feedback regulation by retinoic acid (RA). However, the regulation of BCM gene expression in various other tissues is still unknown. In the present study, we identified the full-length cDNA encoding the rat BCM gene and investigated the regulation of its expression in several tissues by RA in the presence of vitamin A deficiency. We cloned the full-length cDNA encoding BCM gene from a rat intestinal cDNA library by hybridization screening. The BCM gene expression was examined using Northern blotting and reverse transcription-PCR analysis. We also investigated whether BCM gene expression was regulated by retinoids in several tissues of vitamin A-deficient rats. Sequence analysis of this clone revealed an open reading frame of 1,701 bases encoding a protein of 566 amino acids. The predicted polypeptide showed 94%, 81%, and 66% identity with mouse, human, and chicken BCM, respectively. Rat BCM mRNA was highly expressed in the intestine and liver, while there was weak expression in the testes, kidneys, and lungs. Immunoblotting revealed that rat BCM is a 64-kDa protein. BCM gene expression was increased in the small intestine by vitamin A deficiency compared with that in rats on a control diet, while this upregulation was suppressed by all-trans RA (ATRA) or 9-cis RA (9-cis RA). BCM gene expression in the lungs and testes was also suppressed by ATRA or 9-cis RA in rats with vitamin A deficiency. However, hepatic BCM gene expression was only decreased by ATRA and renal expression was not affected by either retinoid. As the small intestine is the major site of beta-carotene conversion, intestinal BCM gene expression may be more tightly regulated. These data suggest that BCM gene expression in several tissues may be down-regulated by RA at the level of conversion of beta-carotene to retinal. To prevent an excess of retinol, homeostasis may occur at the level of conversion of beta-carotene to retinal in several tissues.

Vitamin A Formation in Animals: Molecular Identification and Functional Characterization of Carotene Cleaving Enzymes

Vitamin A and its derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation. It has been known for 70 y that the key step in vitamin A biosynthesis is the oxidative cleavage of a carotenoid with provitamin A activity. While a detailed biochemical characterization of the respective enzymes could be achieved in cell-free homogenates, their molecular nature has remained elusive for a long time. Recent research led to the identification of genes encoding two different types of carotene oxygenases from animal species. The molecular cloning of these different types of animal carotene oxygenases establishes the existence of a family of carotenoid metabolizing enzymes in animals heretofore described in plants. With these tools in hands, old questions in vitamin A research can be definitively addressed on the molecular levels contributing to a mechanistic understanding of the regulation of vitamin A homeostasis or tissue specificity of vitamin A formation, with impact on animal physiology and human health.

Functional rescue of vitamin C synthesis deficiency in human cells using adenoviral-based expression of murine l-gulono-γ-lactone oxidase

l-Gulono-γ-lactone oxidase (GULO) is a critical enzyme present in most mammalian species that is required for the terminal step in vitamin C biosynthesis. Primates are absolutely dependent on exogenously supplied dietary vitamin C due to inactivation of the Gulo gene by mutation over 40 million years ago. In this study, we report the cloning and expression of the murine l-gulono-γ-lactone oxidase cDNA and gene. The cDNA (2.3 kb) encodes an open reading frame of 440 amino acids that shows high homology to the rat l-gulono-γ-lactone oxidase (>94%). The Gulo gene is 22 kb long and contains 12 exons. The 11 introns range in size from 479 to 5641 bp. Northern blot analysis revealed high expression of Gulo transcript in the liver. To investigate whether metabolic loss of vitamin C biosynthesis in human cells can be corrected by heterologous expression of GULO, we constructed a first-generation adenoviral vector expressing the murine GULO cDNA under the transcriptional control of the murine cytomegalovirus (MCMV) early promoter. Low rescue efficiency of Gulo-expressing adenoviral constructs and reduced viral growth in HEK293 cells were observed, suggesting that overexpression of Gulo may be inhibitory to cell growth. Placement of a removable stuffer fragment flanked by lox sites between the MCMV promoter and the Gulo gene resulted in efficient vector rescue and normal viral replication in parental HEK293 cells and high-level expression of Gulo in HEK293 cells expressing Cre recombinase. Cells infected with Gulo-expressing vectors overexpressed an FAD-containing protein that corresponded in size to that predicted for recombinant GULO protein and expressed a functional enzyme as measured by the conversion of l-gulono-γ-lactone to ascorbic acid in cell-free extracts. The cloning of the murine Gulo cDNA and the construction of Gulo-expressing adenoviral vectors are vital steps toward determining the role of vitamin C in basic metabolism and in disease.

Identification of beta-carotene 15, 15'-monooxygenase as a peroxisome proliferator-activated receptor target gene.

Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.

Crystal Structure of Human Riboflavin Kinase Reveals a β Barrel Fold and a Novel Active Site Arch

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B 2) to form FMN, an obligatory step in vitamin B 2 utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel β barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3 10 helix, and a reverse turn followed by a short β strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.

Expression of beta-carotene 15,15' monooxygenase in retina and RPE-choroid.

Beta-carotene 15,15' monooxygenase (beta-CM) catalyzes the central cleavage of beta-carotene to all-trans-retinal, the first step in vitamin A synthesis. This study was conducted to determine the expression of beta-CM in the mammalian retina and RPE, to assess its relevance in carotenoid-retinoid metabolism in the retina and RPE. RT-PCR was used to detect expression of beta-CM mRNA in the retina and RPE-choroid of the mouse, cow, human, and monkey and in RPE cells and other cell lines. Immunofluorescence microscopy was used to localize beta-CM in mouse and monkey retina with an anti-peptide antibody specific for beta-CM. By RT-PCR, beta-CM mRNA was detected at a low level in mouse and monkey retina and in the RPE-choroid of the monkey but not of the mouse. Conversely, beta-CM mRNA was expressed at a low level in both human and bovine RPE-choroid, but not in the retina of either. RPE primary cultured cells of the monkey also showed beta-CM mRNA expression, although the three human lines did not. In addition, of nine other cell lines tested, only COS-7 was positive for beta-CM. Immunofluorescence microscopy showed weak immunoreactivity in the inner retina in both the mouse and monkey. beta-CM immunoreactivity was not detectable in RPE of the mouse. Use of a long-wavelength exciting and emitting secondary probe to mitigate lipofuscin autofluorescence, facilitated the detection of a low level of beta-CM immunoreactivity in monkey RPE. Beta-CM mRNA and protein are expressed at low levels in the mammalian retina and RPE-choroid. Given the low and variable expression of beta-CM in the retina and RPE, it can be concluded that beta-CM is not necessary for a conserved retina or RPE-specific function, but may be necessary for a species-specific function.

Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.

Megalin is an endocytic receptor highly expressed in the proximal tubules of the kidney. Recently, we demonstrated that this receptor is essential for the renal uptake and conversion of 25-OH vitamin D3 to 1,25-(OH)2 vitamin D3, a central step in vitamin D and bone metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded the detailed analysis of the significance of megalin for calcium homeostasis and bone turnover in vivo. Here, we have generated a new mouse model with conditional inactivation of the megalin gene in the kidney by using Cre recombinase. Animals with a renal-specific receptor gene defect were viable and fertile. However, lack of receptor expression in the kidney results in plasma vitamin D deficiency, in hypocalcemia and in severe bone disease, characterized by a decrease in bone mineral content, an increase in osteoid surfaces, and a lack of mineralizing activity. These features are consistent with osteomalacia (softening of the bones) as a consequence of hypovitaminosis D and demonstrate the crucial importance of the megalin pathway for systemic calcium homeostasis and bone metabolism.

Identification and Characterization of a Mammalian Enzyme Catalyzing the Asymmetric Oxidative Cleavage of Provitamin A

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation invivo

Visual pigments (rhodopsins) are composed of a chromophore (vitamin A derivative) bound to a protein moiety embedded in the retinal membranes. Animals cannot synthesize the visual chromophore de novo but rely on the uptake of carotenoids, from which vitamin A is formed enzymatically by oxidative cleavage. Despite its importance, the enzyme catalyzing the key step in vitamin A formation resisted molecular analyses until recently, when the successful cloning of a cDNA encoding an enzyme with beta,beta-carotene-15,15'-dioxygenase activity from Drosophila was reported. To prove its identity with the key enzyme for vitamin A formation in vivo, we analyzed the blind Drosophila mutant ninaB. In two independent ninaB alleles, we found mutations in the gene encoding the beta,beta-carotene-15,15'-dioxygenase. These mutations lead to a defect in vitamin A formation and are responsible for blindness of these flies.

Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation invivo.

Visual pigments (rhodopsins) are composed of a chromophore (vitamin A derivative) bound to a protein moiety embedded in the retinal membranes. Animals cannot synthesize the visual chromophore de novo but rely on the uptake of carotenoids, from which vitamin A is formed enzymatically by oxidative cleavage. Despite its importance, the enzyme catalyzing the key step in vitamin A formation resisted molecular analyses until recently, when the successful cloning of a cDNA encoding an enzyme with beta,beta-carotene-15,15'-dioxygenase activity from Drosophila was reported. To prove its identity with the key enzyme for vitamin A formation in vivo, we analyzed the blind Drosophila mutant ninaB. In two independent ninaB alleles, we found mutations in the gene encoding the beta,beta-carotene-15,15'-dioxygenase. These mutations lead to a defect in vitamin A formation and are responsible for blindness of these flies.

Solvent Regeneration in the Synthesis of Vitamin E

For most drugs, development of the optimum production technologies is determined by the correct choice of solvents and solvent regeneration methods. In particular, this is valid for the final step of vitamin E production: the stage of -tocopherol synthesis and acetylation with acetic anhydride. During the more than half a century that passed since Karrer and Fritzsche [1] reported on the synthesis of vitamin E, a number of solvents were tried in both commercial and laboratory practice. These include aromatic hydrocarbons (benzene, toluene, xylene), aliphatic hydrocarbons (heptane, octane, nonane), alkane derivatives (chloro, nitro, etc.), organic acids, ketones, and esters. Among the special requirements to the solvents employed in this particular process, important points are a not too high boiling temperature (about 100°C), large solvating capacity, ability to dissolve all initial reagents (including catalyst in a homogeneous catalytic process), ability of water removal from the reaction zone in the form of an azeotrope, possibility of washing the catalyst, and small solvent losses in the course of regeneration. Acetic acid esters meet all these requirements to a maximum extent. However, the butyl acetate employed at present in the commercial processes boils at a relatively high temperature (125°C), which favors the formation of side products contaminating vitamin E under the commercial synthesis conditions. A highly promising solvent for the commercial synthesis of vitamin E is ethyl acetate (EA). Indeed, EA possesses a very good solvating capacity with respect to the reagent (trimethylhydroquinone) and allows the process temperature to be reduced to 80°C, which would certainly increase the product (vitamin E) quality. However, there are some problems related to the regeneration of ethyl acetate: in comparison with butyl acetate, EA is more soluble in water, more volatile, and more subject to hydrolysis under the conditions of synthesis. Previously [2], we suggested a wasteless scheme for the regeneration of EA, which allowed this solvent to be multiply reused in the synthesis of vitamin E. The scheme involves four rectification columns and one reaction-rectification column characterized by high reflux numbers. However, a considerable increase in the cost of energy has posed a problem of developing an energy-saving EA regeneration technology. Here we propose an improved regeneration scheme, which is based on the chemical interaction of acetic anhydride in the reaction-rectification column with ethanol and water present in the used EA. This technological scheme provi

Biosynthesis of vitamin B 12: the multi-enzyme synthesis of precorrin-4 and factor IV

Background: In order to study the biosynthesis of vitamin B 12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. Results: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl- l-methionine were included in the incubation, the product was precorrin-4. Both precorrin-3B and precorrin-4 are known precursors of vitamin B 12 and their availability has allowed new mechanistic studies of enzymic transformations. Conclusions: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B 12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B 12 biosynthesis.

Retinyl ester hydrolytic activity associated with human intestinal brush border membranes

Hydrolysis of retinyl esters in the lumen of the small intestine is required before absorption. Previously, rat brush border membranes (BBMs) were found to contain a pancreatic-derived esterase preferring retinyl esters with short fatty acyl chains and an intrinsic esterase preferring esters with long fatty acyl chains. Here, similar activities were found for preparations of human BBMs. Long-chain ester hydrolysis was stimulated best by deoxycholate, a dihydroxy bile salt, whereas short-chain ester hydrolysis was stimulated best by taurocholate, a trihydroxy bile salt. A 10-fold difference in KM values for retinyl palmitate (0.53 mumol/L) and caproate (5.5 mumol/L) also indicated distinguishable long-chain and short-chain activities. Differences between retinyl butyrate and retinyl caproate hydrolysis suggested the possible presence of two short-chain esterase activities associated with both rat and human BBMs, in addition to the long-chain activity. Similarities between human BBM retinyl ester hydrolytic activities and those observed for rat BBMs suggest that the rat is a good model for humans in this step of vitamin A metabolism.

A possible genetic defect in 25-hydroxylation as a cause of rickets

We examined two siblings who had severe rickets at ages 2 and 7 years, respectively, despite a history of adequate vitamin D intake. The patients' sera had calcium concentrations at the lower limits of normal, low phosphate concentrations, elevated alkaline phosphatase activity, and low levels of 25-hydroxyvitamin D. Treatment with high doses of vitamin D2 resulted in resolution of the biochemical abnormalities and radiographic deformities; pharmacologic doses of vitamin D2 were required to maintain normal concentrations of 25-hydroxyvitamin D in the serum even though vitamin D absorption was normal. These children may have a genetic defect of the 25-hydroxylation step in vitamin D activation.