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.

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.

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.

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.

L-Arabinosuria: a new defect in human pentose metabolism.

A female patient, the first child of healthy non-consanguineous parents, presented at the age of 16 months with delayed motor development and facial dysmorphism. In addition she displayed a palatoschizis and multiple skeletal abnormalities as hypoplastic scapulae, hypoplastic os ilea, and an extreme cervical kyphosis. Biochemical investigation of urine revealed no abnormalities except for the presence of large amounts of reducing sugars. The sugar was identified as L-arabinose, which mainly originated from fruit formula in her diet. In addition highly elevated levels of L-arabitol were found in urine, plasma, and cerebrospinal fluid. Although little is known about human arabinose metabolism, we presume that L-arabitol dehydrogenase is deficient in our patient. As polyols are potentially toxic to the central nervous system there could be deleterious long-term effects of this disorder. Withdrawal of dietary fruit led to normalization of polyol levels. The above-mentioned clinical abnormalities are probably not related to this new inborn error of metabolism and should be considered as a separate entity.

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.

The L-gulono-gamma-lactone oxidase gene (GULO) which is a candidate for vitamin C deficiency in pigs maps to chromosome 14.

Vitamin C deficient pigs, when fed a diet lacking L-ascorbic acid (AscA), manifest deformity of the legs, multiple fractures, osteoporosis, growth retardation and haemorrhagic tendencies. This trait was shown by others to be controlled by a single autosomal recessive allele designated as od (osteogenic disorder). The inability of AscA biosynthesis in primates and guinea pigs that exhibit similar symptoms, when they are not supplemented with AscA in the food, was traced to the lack of L-gulono-gamma-lactone oxidase, which catalyzes the terminal step in the biosynthesis of AscA. The non-functional GULOP was mapped to human chromosome 8p21 that corresponds to an evolutionarily conserved segment on either porcine chromosome 4 (SSC4) or 14 (SSC14). We investigated linkage between OD and SSC4- and 14-specific microsatellite loci in order to map the OD locus. Twenty-seven informative meioses in families from one sire and three dams revealed linkage of od with microsatellites SW857 and S0089, located in the subcentromeric region of SSC14. We isolated part of the GULO gene of the pig by screening a porcine genomic library using a pig GULO cDNA as a probe, and mapped it to SSC14q14 by fluorescence in situ hybridization (FISH). Thus, the porcine GULO gene is both a good physiological and positional candidate gene for vitamin C deficiency in pigs.

An animal cell mutant with a deficiency in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Effects on the biosynthesis of ether-linked and diacyl glycerolipids.

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA. K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO+ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-3H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with 32Pi showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA. K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.

Functional assessment of proliferating hepatocytes stimulated by hepatic stimulatory substance in ascorbic acid biosynthetic enzyme-deficient rats.

The functional ability of hepatic stimulatory substance (HSS)-stimulated proliferating hepatocytes was investigated by intrasplenic and/or intraportal transplantation in ascorbic acid (AsA) biosynthetic enzyme-deficient (ODS-od/od) rats that die of osteogenic disorders unless there is AsA supplementation. HSS was extracted from regenerating porcine livers. Hepatocytes isolated from the livers of congeneic ODS-+/+ rats that are capable of synthesizing AsA were transplanted into the spleen (Sp-HTx) and/or the portal vein (Pv-HTx) of ODS-od/od rats. The recipients were divided into eight groups as follows: HSS-untreated groups [group Ia, sham-operated, HTx(-); group IIa, Sp-HTx; group IIIa, Pv-HTx; and group IVa, Sp- and Pv-HTx], HSS-treated groups [group Ib, HSS only; group IIb, Sp-HTx + HSS; group IIIb, Pv-HTx + HSS; and group IVb, Sp- and Pv-HTx + HSS]. The recipients were given a diet and water containing AsA for 6 weeks after HTx, and AsA supplementation was then halted. The average bromodeoxyuridine (BrdU) labeling index (LI) and hepatocyte-occupied ratio in the spleen (H/S ratio) of HSS-treated rats were significantly higher than those of HSS-untreated rats. All the rats in HSS-untreated groups and group Ib died by 8 weeks after the cessation of AsA. In HSS-treated groups IIb, IIIb, and IVb, the survival rates were 60%, 50%, and 80%, respectively, at 16 weeks after HTx. The average serum AsA level of the surviving rats in groups IIb, IIIb, and IVb was significantly higher than that in HSS-untreated groups. These results indicate that HSS treatment induced rapid proliferation of transplanted hepatocytes in the spleen and the portal vein, and that these proliferating hepatocytes synthesized AsA and improved the survival rate of ODS-od/ od rats.

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.

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.

D-arabitol metabolism in Candida albicans: construction and analysis of mutants lacking D-arabitol dehydrogenase.

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol + NAD <=> D-ribulose + NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol or D-arabinose medium. Thus, ArDH catalyzes the first step in D-arabitol utilization and a necessary intermediate step in D-arabinose utilization. Unexpectedly, the ard null mutant synthesized D-arabitol from glucose. Moreover, 13C nuclear magnetic resonance studies showed that the ard null mutant and its wild-type parent synthesized D-arabitol via the same pathway. These results imply that C. albicans synthesizes and utilizes D-arabitol via separate metabolic pathways, which was not previously suspected for fungi.

[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.