Development of a rapid UPLC-MS/MS determination of urine sulfocysteine for diagnosis of sulfocysteinuria and molybdenum co-factor deficiencies.

AIM: Molybdenum co-factor deficiencies and isolated sulfite oxidase deficiency are rare autosomal recessively inherited diseases characterized by severe psychomotor impairment, intractable seizures, dislocated lens and dysmorphic facial features. The biochemical diagnosis of these diseases requires the determination of urine sulfocysteine. MATERIALS & METHODS: Urine sulfocysteine was quantified by an ultra-high performance liquid chromatography-MS/MS assay. The method was validated for linearity, accuracy, precision, recovery and stability. RESULTS & CONCLUSION: Total imprecision of accuracy was less than 6%. Intra-assay and inter-assay precisions were less than 5%. The recovery was higher than 98%. The method is inexpensive, fast, accurate and has been successfully used for identifying five molybdenum co-factor deficient and six sulfite oxidase deficient patients since deployed.

Nutrient deficiencies modify the ionomic composition of plant tissues: a focus on cross-talk between molybdenum and other nutrients in Brassica napus.

The composition of the ionome is closely linked to a plant's nutritional status. Under certain deficiencies, cross-talk induces unavoidable accumulation of some nutrients, which upsets the balance and modifies the ionomic composition of plant tissues. Rapeseed plants (Brassica napus L.) grown under controlled conditions were subject to individual nutrient deficiencies (N, K, P, Ca, S, Mg, Fe, Cu, Zn, Mn, Mo, or B) and analyzed by inductively high-resolution coupled plasma mass spectrometry to determine the impact of deprivation on the plant ionome. Eighteen situations of increased uptake under mineral nutrient deficiency were identified, some of which have already been described (K and Na, S and Mo, Fe, Zn and Cu). Additionally, as Mo uptake was strongly increased under S, Fe, Cu, Zn, Mn, or B deprivation, the mechanisms underlying the accumulation of Mo in these deficient plants were investigated. The results suggest that it could be the consequence of multiple metabolic disturbances, namely: (i) a direct disturbance of Mo metabolism leading to an up-regulation of Mo transporters such as MOT1, as found under Zn or Cu deficiency, which are nutrients required for synthesis of the Mo cofactor; and (ii) a disturbance of S metabolism leading to an up-regulation of root SO42- transporters, causing an indirect increase in the uptake of Mo in S, Fe, Mn, and B deficient plants.

Molybdenum deprivation, purine ingestion and an astrocyte-associated motor neurone syndrome in sheep: assumed clinical effects of inosine.

BACKGROUND: An astrocyte-associated motor neurone syndrome was produced in molybdenum-deprived sheep fed xanthosine. Mo-deprived sheep fed inosine, adenosine or guanosine would be also expected to develop astrocyte-associated motor neurone syndromes, because all these purine nucleosides can act as neuromodulators and all depend on the Mo-associated enzyme xanthine oxidase-dehydrogenase for their catabolism. DESIGN: To investigate the relationship between inosine ingestion and low Mo concentration, eight sheep were fed lucerne chaff with a Mo value <0.10 ppm and the Mo antagonist, sodium tungstate, for 21 weeks, with inosine (35 mg/kg/day) fed for the last 18 of these weeks. This clinical study was uncontrolled. RESULTS: An astrocyte-associated motor neurone syndrome was produced in three sheep 18-27 months later. It was characterised by diaphragmatic, laryngeal, lingual and pharyngeal muscle weakness. The diaphragmatic muscle weakness was the most severe and potentially lethal. CONCLUSION: These findings suggest that purinergic neuromodulation of respiration, vocalisation and swallowing is different to that of limb movement. The syndrome produced, and assumed to be caused by the treatment given, has not been reported in livestock. A similar syndrome is seen in human motor neurone disease, but not in equine motor neurone disease, and this is consistent with it being an upper, not a lower, motor neurone effect.

Low molybdenum state induced by tungsten as a model of molybdenum deficiency in rats.

Organ molybdenum (Mo) concentration and the activity of hepatic sulfite oxidase and xanthine oxidase were compared in tungsten-administered rats as well as rats fed with a low Mo diet to evaluate the use of tungsten-administered rats as a model of Mo deficiency. Twenty-four male 6-week-old Wistar rats were divided into four groups according to diet (AIN93G diet (control diet) or the control diet minus ammonium molybdate (low Mo diet)) and drinking water (deionized water or deionized water containing 200 µg/mL tungsten in the form of sodium tungstate). Mo content in the control and low Mo diets were 196 and 42 ng/g, respectively. Intake of the low Mo diet significantly reduced the Mo content of several organs and serum. Decrease in hepatic sulfite oxidase activity was also induced by the low Mo diet. The administration of tungsten induced marked decreases in organ Mo content and the activity of hepatic sulfite oxidase and xanthine oxidase. These decreases induced by tungsten administration were more pronounced than those induced by just a low Mo diet. Serum uric acid was also reduced by tungsten administration irrespective of Mo intake. Although a comparatively high accumulation of tungsten (3 to 9 µg/g) was observed in the kidneys and liver, adverse effects of tungsten accumulation on liver and kidney function were not observed in serum biochemical tests. These results indicate that tungsten-administered animals may be used as a model of Mo deficiency.

Motor neurone disease in molybdenum-deficient sheep fed the endogenous purine xanthosine: possible mechanism for Tribulus staggers.

BACKGROUND: The occurrence of Tribulus terrestris motor neurone disease (MND) in sheep is linked with grazing Tribulus growing on cultivation paddocks. A previous survey found that the molybdenum (Mo) content of Tribulus growing on uncultivated soils in the Coonabarabran district of New South Wales was 3.03 ppm, but on cultivated soils it was <0.04 ppm. Tribulus contains the purine, xanthosine, which functions as a neuromodulator, and the catabolism of xanthosine is Mo-dependent. DESIGN: To investigate the relationship between xanthosine ingestion and low Mo concentration, eight sheep were fed Mo-deficient lucerne chaff (<0.10 ppm), the Mo antagonist, sodium tungstate, and xanthosine (25 mg/kg/day) over 18 weeks and then returned to pasture. RESULTS: Signs of MND developed in two sheep 30 months later and astrocyte degeneration occurred in all sheep. CONCLUSION: The findings were similar to those observed in sheep with T. terrestris MND, suggesting that the combination of xanthosine ingestion and Mo deficiency may be the cause of this disorder.

Role of trace elements in parenteral nutrition support of the surgical neonate.

BACKGROUND: Parenteral nutrition (PN) has transformed the outcome for neonates with surgical problems in the intensive care unit. Trace element supplementation in PN is a standard practice in many neonatal intensive care units. However, many of these elements are contaminants in PN solutions, and contamination levels may, in themselves, be sufficient for normal metabolic needs. Additional supplementation may actually lead to toxicity in neonates whose requirements are small. METHODS: An electronic search of the MEDLINE, Cochrane Collaboration, and SCOPUS English language medical databases was performed for the key words "trace elements," "micro-nutrients," and "parenteral nutrition additives." Studies were categorized based on levels of evidence offered, with randomized controlled trials and meta-analyses accorded the greatest importance at the apex of the data pool and case reports and animal experiments the least importance. Articles were reviewed with the primary goal of developing uniform recommendations for trace element supplementation in the surgical neonate. The secondary goals were to review the physiologic role, metabolic demands, requirements, losses, deficiency syndromes, and toxicity symptoms associated with zinc, copper, chromium, selenium, manganese, and molybdenum supplementation in PN. RESULTS: Zinc supplementation must begin at initiation of PN. All other trace elements can be added to PN 2 to 4 weeks after initiation. Copper and manganese need to be withheld if the neonate develops PN-associated liver disease. The status of chromium supplementation is currently being actively debated, with contaminant levels in PN being sufficient in most cases to meet neonatal requirements. Selenium is an important component of antioxidant enzymes with a role in the pathogenesis of neonatal surgical conditions such as necrotizing enterocolitis and bronchopulmonary dysplasia. Premature infants are often selenium deficient, and early supplementation has shown a reduction in sepsis events in this age group. CONCLUSION: Appropriate supplementation of trace elements in surgical infants is important, and levels should be monitored. In certain settings, it may be more appropriate to individualize trace element supplementation based on the predetermined physiologic need rather than using bundled packages of trace elements as is the current norm. Balance studies of trace element requirements should be performed to better establish clinical recommendations for optimal trace element dosing in the neonatal surgical population.

Biology of the molybdenum cofactor.

The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.

Mineral deficiencies in tule elk, Owens Valley, California.

Male tule elk (Cervus elaphus nannodes) are susceptible to high rates of antler breakage in Owens Valley, California. We hypothesized that a mineral deficiency in the diet predisposed male elk to antler breakage. We analyzed elk antler, liver, and forage samples to identify mineral imbalances. We compared the mineral content of livers and antlers from elk in Owens Valley to samples taken from tule elk at Grizzly Island Wildlife Area, a population experiencing normal rates (<5%) of antler breakage. Antler and liver samples were collected from 1989 to 1993, and in 2002, and were tested for calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), phosphorus (P), sulfur (S), and zinc (Zn). Mineral levels from antler and liver samples were compared to reference values established for elk and deer. We also compared the mineral content of elk forage in Owens Valley, collected in 2002-03, to dietary reference values established for cattle. In antlers, Ca, Fe, and Mg levels were higher in Owens Valley elk than in Grizzly Island elk, although all mineral levels were lower than reference values established for deer antlers. In liver samples, Cu levels from elk in Owens Valley were lower than those from Grizzly Island and lower than minimum reference values; liver Ca and Mo levels were higher in elk from Owens Valley than in those from Grizzly Island. Compared to reference values, elk forage in Owens Valley had high levels of Ca and Mo, and low levels of Cu, P, and Zn. Mineral analyses from antlers, livers, and forage suggest that tule elk in the Owens Valley are Cu and/or P deficient. High levels of Mo and Ca may exacerbate Cu and P deficiencies, respectively. Bone fragility is a symptom of both deficiencies, and an imbalance in Cu, P, or a combination of both, may predispose male tule elk in the Owens Valley to antler breakage.

Response of inoculated foliar fed pea plants (Pisum sativum L.) to reduced Mo supply.

The application of nutrients to the roots and leaves of inoculated pea plants grown under conditions of reduced Mo supply was studied. Pea plants (Pisum sativum L.) were grown on liquid nutrient solution excluding Mo from the media until the 35th day under glasshouse conditions. Plants were inoculated with the bacterial suspension of Rhizobium leguminosarum Bv. Vicae, strain D293 at approximately 10(8) cells per cm3. The foliar fertilizer Agroleaf was applied at 0.3% concentration. Changes in the root nodulation and the activities of the enzymes connected with nitrogen assimilation pathway (nitrate reductase--NR-NADH: EC 1.6.6.1; glutamine synthetase--GS: EC 6.3.1.2; glutamate synthase--NADH-GOGAT: EC 1.4.1.14 and nitrogenase--NG: EC 1.7.99.2) were observed. It was established that the foliar application of nutrients reduced the inhibitory effect on the root nodulation and nitrogen assimilatory enzyme activities due to the Mo shortage.

Molybdenum kinetics in men differ during molybdenum depletion and repletion.

In this study we developed an expanded compartmental model of molybdenum (Mo) kinetics to determine rates of molybdenum distribution during molybdenum depletion and repletion. The model was based on a clinical study in which 4 men consumed a low-molybdenum diet of 22 microg/d (0.23 micromol/d) for 102 d, followed by a high molybdenum diet of 467 microg/d (4.9 micromol/d) for 18 d. Stable isotopes 100Mo and 97Mo were administered orally and intravenously, respectively, at several time points during the study, and serial samples of plasma, urine, and feces were analyzed for 100Mo, 97Mo, and total Mo. Based on plasma, urine, and fecal molybdenum levels, kinetic parameters of distribution and elimination were determined. The rates of molybdenum distribution and elimination were different during depletion and repletion. During high intake, urinary molybdenum excretion was greater than during low intake. In addition, fractional tissue storage of molybdenum was lower during high intake than during low intake. This suggests that low intake results in an adaptation to conserve body Mo, and that high intake results in an adaptation to eliminate Mo. The model also suggested that food-bound molybdenum was approximately 16% less bioavailable than purified Mo. Finally, under the conditions of this study, the model suggested that an intake of 43 microg/d (0.45 micromol/d) would be sufficient to maintain plasma molybdenum levels at steady state. This is a minimum estimate because subjects in this study were in a molybdenum-sparing state. These findings provide an understanding of the adaptations in molybdenum metabolism that take place during depletion and repletion.

The role of molybdenum in agricultural plant production.

BACKGROUND: The importance of molybdenum for plant growth is disproportionate with respect to the absolute amounts required by most plants. Apart from Cu, Mo is the least abundant essential micronutrient found in most plant tissues and is often set as the base from which all other nutrients are compared and measured. Molybdenum is utilized by selected enzymes to carry out redox reactions. Enzymes that require molybdenum for activity include nitrate reductase, xanthine dehydrogenase, aldehyde oxidase and sulfite oxidase. SCOPE: Loss of Mo-dependent enzyme activity (directly or indirectly through low internal molybdenum levels) impacts upon plant development, in particular, those processes involving nitrogen metabolism and the synthesis of the phytohormones abscisic acid and indole-3 butyric acid. Currently, there is little information on how plants access molybdate from the soil solution and redistribute it within the plant. In this review, the role of molybdenum in plants is discussed, focusing on its current constraints in some agricultural situations and where increased molybdenum nutrition may aid in agricultural plant development and yields. CONCLUSIONS: Molybdenum deficiencies are considered rare in most agricultural cropping areas; however, the phenotype is often misdiagnosed and attributed to other downstream effects associated with its role in various enzymatic redox reactions. Molybdenum fertilization through foliar sprays can effectively supplement internal molybdenum deficiencies and rescue the activity of molybdoenzymes. The current understanding on how plants access molybdate from the soil solution or later redistribute it once in the plant is still unclear; however, plants have similar physiological molybdenum transport phenotypes to those found in prokaryotic systems. Thus, careful analysis of existing prokaryotic molybdate transport mechanisms, as well as a re-examination of know anion transport mechanisms present in plants, will help to resolve how this important trace element is accumulated.

New approaches towards laboratory diagnosis of isolated sulphite oxidase deficiency.

BACKGROUND: Molybdenum cofactor deficiency (resulting in combined deficiencies of the enzymes sulphite oxidase, xanthine dehydrogenase and aldehyde dehydrogenase) and isolated sulphite oxidase deficiency are inherited metabolic diseases which follow an autosomal recessive trait of inheritance. Detection of these diseases in selective screening for inborn errors of metabolism is not easy because relevant metabolites are either not routinely determined or are unstable. METHODS: We have searched for additional markers for these diseases and studied plasma total homocysteine (determined by enzyme immunoassay) and S-sulphonation of transthyretin (assessed by electrospray ionization mass spectrometry). RESULTS AND CONCLUSION: We found total homocysteine concentrations below the limit of quantification (<1 micromol/L) in all samples of patients with sulphite oxidase deficiency studied in this regard and that the proportion of S-sulphonated transthyretin is clearly increased in such samples. Our observations suggest additional tools for selective screening and diagnostic work-up of patients suspected of having sulphite oxidase deficiency.

Environmental Se-Mo-B deficiency and its possible effects on crops and Keshan-Beck disease (KBD) in the Chousang area, Yao County, Shaanxi Province, China.

Causes of Keshan-Beck disease (KBD) are still being probed and monitored in China. Relationships between trace elements from eco-environmental systems and KBD are poorly understood although relationships between environmental Se and human health have received extensive attention. In order to investigate relationships between eco-environmental geochemistry and KBD, we selected the Chousang KBD area in Yao County, Shaanxi Province, China, as an example of a prevailing KBD area applying I-Se-rich salts instead of utilizing Se-rich fertilisers on food crops to prevent local residents from developing KBD before 1995. Environmentally geochemical samples (rocks, soils, plants and children's hair) were collected from the Chousang KBD area. Soils in the study area contain 0.11 +/- 0.02 microgram Se g-1, 0.75 +/- 0.11 microgram Mo g-1, and 34.5 +/- 1.5 micrograms B g-1 on average, indicating that the study area is a deficient-Se-Mo-B area. Se (0.07 +/- 0.007 microgram g-1), Mo (0.35 +/- 0.09 microgram g-1) and B (3 +/- 0 micrograms g-1) contents are low in wheat and corn used as a daily main food staple of local inhabitants. It is indicated that the study area is deficient in environmental Se-Mo-B for the local residents. Se contents of children's hair from the Yangyuan Elementary School in the study area range from 0.09 to 0.26 microgram Se g-1 with an average of 0.165 +/- 0.05 microgram Se g-1 (n = 10) in this KBD endemic area. Due to the low levels of Se, Mo and B available in soils and rocks, crops including wheat and corn are deficient in these elements, accordingly, the deficiency of Se, Mo and B in this area may be linked to the daily consumption of wheat and corn deficient in Se, Mo and B. Therefore, local inhabitants should be encouraged to fertilise mixtures of Se, Mo and B on crop plants in order to avoid development of KBD and guarantee a good harvest of crops.

A mutation in the gene for the neurotransmitter receptor-clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency.

Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABA(A). A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo). Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis. Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood. Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin. The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency. Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation.

The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase.

Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent. In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.

Prenatal diagnosis and carrier detection for molybdenum cofactor deficiency type A in northern Israel using polymorphic DNA markers.

Molybdenum cofactor deficiency (MoCoD) is an autosomal recessive, fatal neurological disorder, characterized by the combined deficiency of sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase. We have recently reported an excessive occurrence of this fatal disorder among segments of the Arab population in Northern Israel suggesting that the true incidence of MoCoD is probably underestimated in this highly inbred population. This lethal disease can be diagnosed prenatally by assay of sulphite oxidase activity in chorionic villus samples in pregnancies of couples who have had previously affected children (obligatory carriers). However, to date, there is no biochemical assay for carrier detection among the population at risk. Recently we demonstrated the linkage of a MoCoD gene to an 8-cM region on chromosome 6p21.3 in two consanguineous Israeli-Arab unrelated kindreds. The description of the MOCS1 gene that maps to the same region and which carries multiple mutations in MoCoD type A followed this finding. We describe here one additional kindred of Arab-Israeli origin, which is also linked to the MOCS1 locus, and demonstrate the feasibility of prenatal diagnosis and carrier detection using microsatellite markers in selected families when mutations are unknown. A complete correlation between the biochemical and DNA assays was found in a total of six samples (five chorionic villus and one amniocyte culture sample) obtained from the three MoCoD families.

Molybdenum cofactor deficiency-phenotypic variability in a family with a late-onset variant.

In a family with molybdenum cofactor deficiency, the onset in the index case was delayed until 1 year of age, when the patient presented with an episode of lethargy and inconsolable crying culminating in a seizure. By 17 months she showed mild motor delay, regression in language skills, and feeding difficulties. Progressive global deterioration followed, associated with sustained irritability, dystonic posturing, and further seizures, before her condition subsequently plateaued. Low plasma uric acid, raised urinary xanthine and hypoxanthine, and positive urinary sulphite were found, which, coupled with assay of sulphite oxidase activity in cultured fibroblasts, confirmed the diagnosis. A sibling had isolated lens dislocation and an identical biochemical profile. MRI in both children was strikingly abnormal. Molybdenum cofactor deficiency may present as a late-onset variant with considerable phenotypic variability.