Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

1,3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out.

BACKGROUND: 1,3-propanediol (PDO) is a substantially industrial metabolite used in the polymer industry. Although several natural PDO production hosts exist, e.g. Klebsiella sp., Citrobacter sp. and Clostridium sp., the PDO yield on glycerol is insufficient for an economically viable bio-process. Enhancing this yield via strain improvement can be achieved by disconnecting the production and growth pathways. In the case of PDO formation, this approach results in a microorganism metabolizing glycerol strictly for PDO production, while catabolizing a co-substrate for growth and maintenance. We applied this strategy to improve the PDO production with Citrobacter werkmanii DSM17579. RESULTS: Genetic tools were developed and used to create Citrobacter werkmanii DSM17579 ∆dhaD in which dhaD, encoding for glycerol dehydrogenase, was deleted. Since this strain was unable to grow on glycerol anaerobically, both pathways were disconnected. The knock-out strain was perturbed with 13 different co-substrates for growth and maintenance. Glucose was the most promising, although a competition between NADH-consuming enzymes and 1,3-propanediol dehydrogenase emerged. CONCLUSION: Due to the deletion of dhaD in Citrobacter werkmanii DSM17579, the PDO production and growth pathway were split. As a consequence, the PDO yield on glycerol was improved 1,5 times, strengthening the idea that Citrobacter werkmanii DSM17579 could become an industrially interesting host for PDO production.

Garrod's fourth inborn error of metabolism solved by the identification of mutations causing pentosuria.

Pentosuria is one of four conditions hypothesized by Archibald Garrod in 1908 to be inborn errors of metabolism. Mutations responsible for the other three conditions (albinism, alkaptonuria, and cystinuria) have been identified, but the mutations responsible for pentosuria remained unknown. Pentosuria, which affects almost exclusively individuals of Ashkenazi Jewish ancestry, is characterized by high levels of the pentose sugar L-xylulose in blood and urine and deficiency of the enzyme L-xylulose reductase. The condition is autosomal-recessive and completely clinically benign, but in the early and mid-20th century attracted attention because it was often confused with diabetes mellitus and inappropriately treated with insulin. Persons with pentosuria were identified from records of Margaret Lasker, who studied the condition in the 1930s to 1960s. In the DCXR gene encoding L-xylulose reductase, we identified two mutations, DCXR c.583ΔC and DCXR c.52(+1)G > A, each predicted to lead to loss of enzyme activity. Of nine unrelated living pentosuric subjects, six were homozygous for DCXR c.583ΔC, one was homozygous for DCXR c.52(+1)G > A, and two were compound heterozygous for the two mutant alleles. L-xylulose reductase was not detectable in protein lysates from subjects' cells and high levels of xylulose were detected in their sera, confirming the relationship between the DCXR genotypes and the pentosuric phenotype. The combined frequency of the two mutant DCXR alleles in 1,067 Ashkenazi Jewish controls was 0.0173, suggesting a pentosuria frequency of approximately one in 3,300 in this population. Haplotype analysis indicated that the DCXR c.52(+1)G > A mutation arose more recently than the DCXR c.583ΔC mutation.

Vulnerable atherosclerotic plaque morphology in apolipoprotein E-deficient mice unable to make ascorbic Acid.

BACKGROUND: Oxidative stress is thought to play an important role in atherogenesis, suggesting that antioxidants could prevent coronary artery disease. However, the efficacy of vitamin C in reducing atherosclerosis is debatable in humans and has not been tested rigorously in animals. METHODS AND RESULTS: Gulo(-/-)Apoe(-/-) mice were used to test a hypothesis that chronic vitamin C deficiency enhances the initiation and development of atherosclerosis. These mice are dependent on dietary vitamin C because of the lack of L-gulonolactone-gamma-oxidase and are prone to develop atherosclerosis because of lacking apolipoprotein E. Beginning at 6 weeks of age, the Gulo(-/-)Apoe(-/-) mice were fed regular chow or Western-type diets containing high fat and supplemented with either 0.033 g or 3.3 g/L of vitamin C in their drinking water. This regimen produced mice with chronically low vitamin C (average 1.5 microg/mL in plasma) or high vitamin C (average 10 to 30 microg/mL in plasma). Morphometric analysis showed that within each sex, age, and diet group, the sizes of the atherosclerotic plaques were not different between low vitamin C mice and high vitamin C mice. However, advanced plaques in the low vitamin C mice had significantly reduced amounts of Sirius red-staining collagen (36.4+/-2.2% versus 54.8+/-2.3%, P<0.0001), larger necrotic cores within the plaques, and reduced fibroproliferation and neovascularization in the aortic adventitia. CONCLUSIONS: Chronic vitamin C deficiency does not influence the initiation or progression of atherosclerotic plaques but severely compromises collagen deposition and induces a type of plaque morphology that is potentially vulnerable to rupture.

Molecular basis for the deficiency in humans of gulonolactone oxidase, a key enzyme for ascorbic acid biosynthesis.

The inability of humans to synthesize L-ascorbic acid is known to be due to a lack of L-gulono-gamma-lactone oxidase, an enzyme that is required for the biosynthesis of this vitamin. Isolation of a cDNA for rat L-gulono-gamma-lactone oxidase allowed us to study the basic defect underlying this deficiency at the gene level and led to isolation of a human genomic clone related to L-gulono-gamma-lactone oxidase as well as three overlapping clones covering the entire coding region of the rat L-gulono-gamma-lactone oxidase cDNA. Sequence analysis study indicated that the human L-gulono-gamma-lactone oxidase gene has accumulated a large number of mutations since it stopped being active and that it now exists as a pseudogene in the human genome.

Phospholipid hydroperoxides and lipid peroxidation in liver and plasma of ODS rats supplemented with alpha-tocopherol and ascorbic acid.

Forty-five mutant male ODS rats, unable to synthesize ascorbic acid, were fed nine diets containing 5, 50 or 250 mg of vitamin E/kg diet and 150, 300 or 900 mg of vitamin C/kg diet for 21 days. The concentrations of vitamins C and E increased in liver and plasma in relation to the level of these vitamins in the diet. Vitamin C dietary supplementation increased the plasma vitamin E content at low levels of vitamin E intake, supporting the concept of an in vivo synergism between both antioxidant vitamins. Vitamin C, at the dietary levels studied, did not affect the lipid peroxidation. Vitamin E decreased liver and plasma endogenous levels of thiobarbituric acid-reactive substances and liver sensitivity to non-enzymatic lipid peroxidation. This was confirmed by a highly specific assay of lipid hydroperoxides using high performance liquid chromatography with chemiluminescence detection. The hepatic concentration of both phosphatidylcholine and phosphatidylethanolamine hydroperoxides decreased as the vitamin E content of the diet increased. The results show for the first time the capacity of vitamin E to protest against peroxidation of major phospholipids in vivo under basal unstressed conditions.

Effect of ascorbic acid deficiency on primary and reparative dentinogenesis in non-ascorbate-synthesizing ODS rats.

Ascorbic acid is essential to the biosynthesis of collagen, the major organic matrix component of dentine. The ODS rat is a mutant strain of Wistar rat characterized by hereditary lack of L-gulono-gamma-lactone oxidase and thus is unable to synthesize ascorbic acid. ODS rats were given an ascorbic acid-free diet to investigate how ascorbic acid deficiency affects dentine formation in vivo. Histomorphometric analysis on their growing molars and incisors showed a significant reduction in both size and mineral apposition rate of dentine, as revealed by contact microradiography and fluorescent time-marking, respectively. A similar reduction in bone formation was simultaneously demonstrated in the mandible, confirming the previously reported osteopathic effects of ascorbic acid deficiency. When pulp inflammation was induced in lower first molars by making unsealed pulp exposures, specimens from control animals showed continuous deposition of an osteodentine-like tissue in the radicular pulp chamber; this type of mineralized tissue formation was greatly reduced in ascorbic acid-deprived animals. These results indicate that ascorbic acid deficiency hampers dentine formation under both physiological and pathological conditions of the dentine/pulp complex. ODS rats could be useful in investigating in vivo effects of ascorbic acid deficiency on the formation of dentine and other dental mineralized tissues.

[Hepatocellular transplantation in rats with congenital ascorbic acid deficiency].

The purpose of this study was to investigate suitable sites for hepatocyte transplantation in rats with congenital liver enzyme deficiency. Hepatocytes were isolated from ODS-(+)/+ rats, which are congenic to ODS-od/od rats and have hepatic L-gulonolactone oxidase. A total of 1 x 10(7), 1 x 10(7), and 2.5 x 10(6) hepatocytes were respectively transplanted into the peritoneal cavity, spleen, or portal vein of ODS-od/od rats, which are unable to synthesize ascorbic acid (AsA) due to lack of hepatic L-gulonolactone oxidase. After 4 days of oral pretreatment with 0.05% 2-acetylaminofluorene, recipients underwent 70% partial hepatectomy just before transplantation. AsA administration was discontinued at 6 weeks after transplantation. The symptom-free survival rate and the serum AsA level of recipient rats were determined at 6 weeks after discontinuing AsA administration. The symptom-free survival rate of untransplanted rats and recipient rats with intraperitoneal, intrasplenic and intraportal hepatocyte transplantation were 0%, 0%, 60%, and 100%, respectively. The serum AsA levels were 0.20 +/- 0.20 microgram/ml, 0.14 +/- 0.05 microgram/ml, 1.06 +/- 0.26 microgram/ml, and 1.58 +/- 0.61 microgram/ml, respectively. Intrasplenic or intraportal transplantation was able to cure ODS-od/od rats. A subsequent splenectomy study showed that hepatocytes reaching the liver via the splenic vein following intrasplenic hepatocyte transplantation played a major role in this experimental success.

Dicarbonyl/l-xylulose reductase (DCXR): The multifunctional pentosuria enzyme.

Dicarbonyl/L-xylulose reductase (DCXR) is a highly conserved and phylogenetically widespread enzyme converting L-xylulose into xylitol. It also reduces highly reactive α-dicarbonyl compounds, thus performing a dual role in carbohydrate metabolism and detoxification. Enzymatic properties of DCXR from yeast, fungi and mammalian tissue extracts are extensively studied. Deficiency of the DCXR gene causes a human clinical condition called pentosuria and low DCXR activity is implicated in age-related diseases including cancers, diabetes, and human male infertility. While mice provide a model to study clinical condition of these diseases, it is necessary to adopt a physiologically tractable model in which genetic manipulations can be readily achieved to allow the fast genetic analysis of an enzyme with multiple biological roles. Caenorhabditis elegans has been successfully utilized as a model to study DCXR. Here, we discuss the biochemical properties and significance of DCXR activity in various human diseases, and the utility of C. elegans as a research platform to investigate the molecular and cellular mechanism of the DCXR biology.

Mannitol utilisation is required for protection of Staphylococcus aureus from human skin antimicrobial fatty acids.

Mannitol (Mtl) fermentation, with the subsequent production of acid, is a species signature of Staphylococcus aureus, and discriminates it from most other members of the genus. Inactivation of the gene mtlD, encoding Mtl-1-P dehydrogenase was found to markedly reduce survival in the presence of the antimicrobial fatty acid, linoleic acid. We demonstrate that the sugar alcohol has a potentiating action for this membrane-acting antimicrobial. Analysis of cellular metabolites revealed that, during exponential growth, the mtlD mutant accumulated high levels of Mtl and Mtl-P. The latter metabolite was not detected in its isogenic parent strain or a deletion mutant of the entire mtlABFD operon. In addition, the mtlD mutant strain exhibited a decreased MIC for H2O2, however virulence was unaffected in a model of septic arthritis.

Epididymal P34H protein deficiency in men evaluated for infertility.

Our study aimed to compare the frequency of epididymal protein P34H deficiency in a population of men undergoing routine infertility evaluation with that in men with proven fertility. Our results suggest that a significant proportion of men investigated for male infertility may be epididymal protein P34H deficient.

Human L-xylulose reductase variation: family and population studies.

Only one of the two main L-xylulose reductases present in human tissue is deficient in individuals with essential pentosuria (Lane, 1985). The isozyme which is affected by the pentosuria mutation occurs as mitochondrial and cytosolic forms in normal individuals, whereas the other isozyme (which is not affected by the mutation) occurs only in the cytosol. A new assay of red cell L-xylulose reductase activity has facilitated the identification of carriers of the essential pentosuria allele at both family and population levels. Reinvestigation of a Lebanese family in which pentosuria has previously been thought to be dominantly inherited reveals that the condition is recessively inherited in this family as well. A minimum estimate of the frequency of the pentosuria allele in an Ashkenazi-Jewish population, calculated from the apparent heterozygote frequency, is 0.0127. The likelihood of the Ashkenazi and Lebanese pentosuria alleles being the same is discussed.

Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species.

Guinea pigs cannot synthesize L-ascorbic acid because of their deficiency in L-gulono-gamma-lactone oxidase, a key enzyme for the biosynthesis of this vitamin in higher animals. In this study we isolated the L-gulono-gamma-lactone oxidase gene of the rat and the homologue of this gene of the guinea pig by screening rat and guinea pig genomic DNA libraries in lambda phage vectors, respectively, using a rat L-gulono-gamma-lactone oxidase cDNA as a probe. Sequencing analysis showed that the amino acid sequence of the rat enzyme is encoded by 12 exons and that all the intron/exon boundaries follow the GT/AG rule. On the other hand, regions corresponding to exons I and V were not identified in the guinea pig L-gulono-gamma-lactone oxidase gene homologue. Other defects found in this gene homologue are a deletion of the nucleotide sequence corresponding to a 3' 84-base pair part of rat exon VI, a 2-base pair deletion in the remaining exon VI-related region, and nonconformance to the GT/AG rule at one of the putative intron/exon boundaries. Furthermore, a large number of mutations were found in the amino acid-coding regions of the guinea pig sequence; more than half of them lead to nonconservative amino acid changes, and there are three stop codons as well. Thus it is clear that the guinea pig homologue of the L-gulono-gamma-lactone oxidase gene exists as a pseudogene that randomly accumulated a large number of mutations without functional constraint since the gene ceased to be active during evolution. On the basis of the neutral theory of evolution, the date of the loss of L-gulono-gamma-lactone oxidase in the ancestors of the guinea pig was roughly calculated to be less than 20 million years ago.

Screening for red blood cell sorbitol dehydrogenase deficiency in patients with diabetes or cataracts.

Quantitative screening for red blood cell sorbitol dehydrogenase (RBC-SORD) deficiency in 111 patients with juvenile onset diabetes, 92 patients with adult onset diabetes, 42 patients with idiopathic cataracts and 192 professional blood donors was performed. A wide variability in RBC-SORD activity in controls and patients was observed. No significant differences in SORD activity either between patients with diabetes and patients with idiopathic cataracts or between diabetics with and without cataracts were observed. Whether or not there were carriers for either amorphous or hypomorphous alleles of the SORD locus in the population studied could not be defined in terms of enzymatic activity levels.