A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease.

Galactosylceramidase (GALC) is the lysosomal ß-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a ß-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease.

Subclinical zinc deficiency impairs pancreatic digestive enzyme activity and digestive capacity of weaned piglets.

This study investigated the effects of short-term subclinical Zn deficiency on exocrine pancreatic activity and changes in digestive capacity. A total of forty-eight weaned piglets were fed ad libitum a basal diet (maize and soyabean meal) with adequate Zn supply (88 mg Zn/kg diet) during a 2-week acclimatisation phase. Animals were then assigned to eight dietary treatment groups (n 6) according to a complete randomised block design considering litter, live weight and sex. All pigs were fed restrictively (450 g diet/d) the basal diet but with varying ZnSO4.7H2O additions, resulting in 28·1, 33·6, 38·8, 42·7, 47·5, 58·2, 67·8 and 88·0 mg Zn/kg diet for a total experimental period of 8 d. Pancreatic Zn concentrations and pancreatic activities of trypsin, chymotrypsin, carboxypeptidase A and B, elastase and α-amylase exhibited a broken-line response to stepwise reduction in dietary Zn by declining beneath thresholds of 39·0, 58·0, 58·0, 41·2, 47·5, 57·7 and 58·0 mg Zn/kg diet, respectively. Furthermore, carboxypeptidase B and α-amylase activities were significantly lower in samples with reduced pancreatic Zn contents. Coefficients of faecal digestibility of DM, crude protein, total lipids and crude ash responded similarly to pancreatic enzyme activities by declining below dietary thresholds of 54·7, 45·0, 46·9 and 58·2 mg Zn/kg diet, respectively. In conclusion, (1) subclinical Zn deficiency impaired pancreatic exocrine enzymes, (2) this response was connected to pancreatic Zn metabolism and (3) the decline in catalytic activity impaired faecal digestibility already after 1 week of insufficient alimentary Zn supply and very early before clinical deficiency symptoms arise.

The effect of the paraoxonase 1 (PON1) T(-107)C polymorphism on serum PON1 activity in women is dependent on fatty acid intake.

Paraoxonase 1 (PON1) is an enzyme that prevents the peroxidation of lipoprotein and cell membranes. Our hypothesis is that the effect of the PON1 T(-107)C polymorphism on serum PON1 activity in healthy adult women is dependent on their fatty acid intake profile. This study included women (n = 39) who completed a food frequency questionnaire. Fatty acid intake was estimated based on the interview and a nutrient reference table. Blood samples were collected for genotyping and to measure serum PON1 activity. Serum PON1 activity was different among genotypes and was higher for women of the CC genotype (P < .001). Women in the study were categorized in 2 groups according to the median nutrient intake. Overall, there was a difference (P < .05) in serum PON1 activity between the CC and TT genotypes in women ingesting either above or below the median total fat, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, omega 3 (n-3) and omega 6 (n-6; P < .05). However, genotype effects on serum PON1 activity were not observed in women ingesting below the median (15:1) ratio of n-6/n-3 (P > .05) but were observed in women ingesting above the ratio of n-6/n-3 (P < .05). This is partly because women of the CC genotype had decreased PON1 activity when ingesting a lower ratio of n-6/n-3 diet (P < .05), while women of the TT genotype had increased PON1 activity (P < .05). In conclusion, the overall presence of the C allele was associated with increased serum PON1 activity, although a diet with high saturated fatty acid or a low ratio of n-6/n-3 reduced PON1 activity in women with the CC genotype.

A randomized trial of copper supplementation effects on blood copper enzyme activities and parameters related to cardiovascular health.

Marginal copper deficiency, which may affect cardiovascular disease risk, is proposed to occur in many adults in Western industrialized countries. The present study tested the hypothesis that in a group of USA adults, increased copper intake would alter readings for blood copper enzymes and markers relevant to cardiovascular disease risk. Healthy middle aged adults with moderately high cholesterol, were given either placebo or copper supplementation (2 mg copper/day as copper glycinate) for 8 weeks. Blood samples were taken before and after the 8 weeks. Copper, but not placebo, raised activities for two copper enzymes, erythrocyte superoxide dismutase 1 and plasma ceruloplasmin. In contrast, five cardiovascular health related plasma parameters were not changed significantly by copper: C-reactive protein, homocysteine, and cholesterol (total, LDL and HDL). However, changes in erythrocyte superoxide dismutase 1 correlated positively with changes in plasma HDL and negatively with plasma homocysteine. Also, copper lowered mean oxidized LDL values, a result that was statistically significant, but inconsistent. In this test population, increased copper intake raised copper enzyme activities, but did not consistently improve the cardiovascular health measures studied.

Clinical and biochemical characterization of patients with early infantile onset of autosomal recessive GTP cyclohydrolase I deficiency without hyperphenylalaninemia.

Autosomal recessive guanosine triphosphate cyclohydrolase (GTPCH) type I deficiency is characterized by complex neurological dysfunction. Patients are usually diagnosed with hyperphenylalaninemia in newborn screening. We describe two unrelated patients without hyperphenylalaninemia who presented during early infancy with severe motor retardation, hypokinesia, and truncal hypotonia. CSF homovanillic acid and 5-hydroxyindoleacetic acid as well as tetrahydrobiopterin and neopterin were decreased. Diagnosis of recessive GTPCH deficiency was confirmed biochemically, and a novel homozygous mutation was identified in one patient and a compound-heterozygous mutation of GCH1 in the other. Treatment with Levodopa/Carbidopa resulted in striking clinical improvement, with age-appropriate development at follow-up at 6 years. Autosomal recessive GTPCH deficiency should be considered in infants with severe truncal hypotonia even if hyperphenylalaninemia or classical extrapyramidal symptoms are missing. Neurotransmitter analysis followed by enzyme or mutation analysis can confirm the diagnosis, and Levodopa treatment should be started at high-doses.

Effect of Brazil nut supplementation on the blood levels of selenium and glutathione peroxidase in hemodialysis patients.

OBJECTIVE: In patients who have undergone hemodialysis, large amounts of reactive oxygen species (ROS) are produced and, at higher concentrations, ROS are thought to be involved in the pathogenesis of cardiovascular disease. It has been proposed that selenium (Se) may exert an antiatherogenic influence by reducing oxidative stress. The richest known food source of selenium is the Brazil nut (Bertholletia excelsa, family Lecythidaceae), found in the Amazon region. We evaluated the effect of Brazil nut supplementation on blood levels of Se and glutathione peroxidase (GSH-Px) activity in patients on hemodialysis. METHODS: A total of 81 patients on hemodialysis (52.0±15.2 y old, average time on dialysis 82.3±91.4 mo, body mass index 24.9±4.4 kg/m(2)) from the RenalCor and RenalVida Clinics in Rio de Janeiro, Brazil, were studied. All patients received one nut (around 5 g, averaging 58.1 µg Se/g) a day for 3 mo. The Se concentrations in the nuts and in plasma and erythrocytes were determined by atomic absorption spectrophotometry with hydride generation (Hitachi, Z-500). GSH-Px levels were measured using Randox commercial kits. RESULTS: Plasma Se (18.8±17.4 µg/L) and erythrocyte (72.4±37.9 µg/L) levels were below the normal range before nut supplementation. After supplementation, the plasma level increased to 104.0±65.0 µg/L and erythrocytes to 244.1±119.5 µg/L (P<0.0001). The activity of GSH-Px also increased after supplementation, from 46.6±14.9 to 55.9±23.6 U/g of hemoglobin (P<0.0001). Before supplementation, 11% of patients had GSH-Px activity below the normal range (27.5-73.6 U/g of hemoglobin). After supplementation, all patients showed GSH-Px activity within the normal range. CONCLUSION: The data revealed that the investigated patients presented Se deficiency and that the consumption of only one Brazil nut a day (5 g) during 3 mo was effective to increase the Se concentration and GSH-Px activity in these patients, thus improving their antioxidant status.

Methionine-deficient diet induces post-transcriptional downregulation of cystathionine ß-synthase.

OBJECTIVE: Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transsulfuration pathway by the action of cystathionine ß-synthase (CBS). We have examined the metabolic response in mice of a shift from a methionine-replete to a methionine-free diet. METHODS AND RESULTS: We found that shifting 3-mo-old C57BL6 mice to a methionine-free diet caused a transient increase in tHcy and an increase in the tHcy/methionine ratio. Because CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 d on the methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicated that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability. CONCLUSION: Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway by an S-adenosylmethionine-independent mechanism that signals a rapid downregulation of CBS protein.

3-phosphoglycerate dehydrogenase deficiency: a case report of a treatable cause of seizures.

Serine deficiency disorders are a new group of neurometabolic diseases resulting from a deficiency in one of the three enzymes in the biosynthetic pathway of L-serine. Deficiency of the enzyme 3-phosphoglycerate dehydrogenase (3-PGDH), which catalyzes the first step in the biosynthetic pathway, leads to congenital microcephaly, severe psychomotor retardation, and intractable seizures. We report a 4 1/2-year-old boy who presented with congenital microcephaly, psychomotor retardation, hypertonia, strabismus, and drug-resistant seizures due to 3-PGDH deficiency. His seizures responded to L-serine and glycine supplementation only. This potentially treatable disease should be borne in mind in patients with congenital microcephaly, psychomotor retardation and seizures. A timely diagnosis based on the detection of low cerebrospinal fluid levels of L-serine and glycine is expected to further increase the success of L-serine and glycine supplementation in these patients.

Pyridoxal 5'-phosphate values in cerebrospinal fluid: reference values and diagnosis of PNPO deficiency in paediatric patients.

Our aim was to establish reference values for cerebrospinal fluid (CSF) pyridoxal 5'-phosphate (PLP) in a paediatric population for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency. For reference values, CSF samples from 113 paediatric controls (age range: 1 day-18 years) from Barcelona and London were analysed. Cerebrospinal fluid PLP and biogenic amine concentrations were analysed by HPLC with fluorescence and electrochemical detection. Pyridoxal 5'-phosphate concentrations in 4 patients with PNPO deficiency were determined. A negative correlation between CSF PLP values and age of controls was observed in both populations (r=-0.503; p<0.0001 and r=-0.542; p=0.002). Reference values were stratified into 4 (Barcelona) and 3 age groups (London). For the newborn period, CSF PLP reference intervals were 32-78 and 44-89 nmol/L for the Barcelona and London centers, respectively). No correlation was observed in the different age groups between PLP values and biogenic amines metabolites. PLP values in neonates with PNPO deficiency were clearly decreased (PLP=3.6, 12.0, 14.0 and 18.0 nmol/L) compared with our reference ranges. In conclusion, reference values for CSF PLP should be stratified according to age. No association was observed between PLP values and biogenic amines metabolites. In our 4 cases with PNPO deficiency, CSF PLP values were clearly below the reference values.

Rationale for diagnosing deficiency of ChEs and for applying exogenous HuChEs to the treatment of diseases.

Recent evidence strongly demonstrates that acetylcholine (ACh) is not only involved in the function of the central and peripheral nervous systems, including the parasympathetic and somatic systems, but also acts as a ubiquitous cell signaling molecule or cytotransmitter, and as a hormone with paracrine, juxtacrine and autocrine properties. This active molecule exerts versatile and potent functions primarily through its specific nicotinic and muscarinic receptors (nAChRs and mAChRs, respectively). These functions modulate numerous biomechanisms, including cell growth, survival, proliferation and differentiation, cell-cell contact, cell cycle, locomotion, electrical activity, immune function, apoptosis, organization of the cytoskeleton, trophic functions, secretion, adhesion, resorption, and stress-response-regulation. By nature, the precise ACh levels and responses from receptors must be controlled and regulated by its degrading enzymes, the cholinesterases (ChEs), namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Once ChEs become critically deficient in quality and quantity, ACh signaling will be uncontrollably aberrant and persistent. An in-depth account of the fundamental roles of ChEs, comprising their diverse soluble and membrane-bound forms, in maintaining the functional equilibrium of ACh in the macro and microenvironment has been undertaken. This work also covers ACh receptors, signaling pathways, other interdependent and interrelated substances, functional processes, role of ChEs as first-line gatekeepers and defenses for the architecture of cells, tissues and organisms, physically, chemically, and structurally. The mechanisms of many diseases ranging from the acute cholinergic crisis to the chronic degenerative and hypergenerative disorders such as Alzheimer's disease, cancers, atopic dermatitis, may involve a deficiency of ChEs or imbalance between ACh and ChEs, initially or consequentially. It is therefore essential to ascertain a ChE deficiency, or an imbalance between ACh and ChEs, in tissues and body fluids in order for conducting clinical diagnosis, prevention and treatment. An argument is put forward on the rationale of applying exogenous human ChEs to reverse enzymatic deficiency and correct the imbalance between ACh and ChEs, to repair the affected receptors and protect against their further loss in the body, and consequently to alleviate the signs and symptoms of diseases. Evidence is adduced for the safety and efficacy of ChEs treatment.

Effect of iodine supplement on iodine status and 5'-deiodinase activity in the brain of neonatal rats with iodine deficiency.

The weanling Wistar rats of iodine deficiency were divided into three groups for supplementation of different levels of iodine (iodine-excessive [IE], iodine-adequate [IA], and iodine-deficient [ID]), with a control group (C). The iodine content in the thyroid was determined by epithermal neutron activation analysis. The activities of 5'-deiodinase and 5-deiodinase in the brains were assayed by determining the conversion ratios of T4 to T3 and rT3, respectively. The thyroid hormones levels in serum were also tested. The results indicated that the ID group had a goiter containing a small amount of iodine, but the IE group had a slightly swollen thyroid with rich iodine; the concentration of iodine per unit mass of thyroid was lower in group IE than in groups IA and C. The highest 5'-deiodinase and lowest 5-deiodinase activities in group ID and the lowest 5'-deiodinase activity in group IE were found. The iodine deficiency or excess resulted in a compensated hypothyroid state. The results suggest that the iodine status and the deiodinases activities would become normal for the rats of iodine deficiency if adequate iodine is supplemented soon after birth. Meanwhile, it is also critical to avoid excessive intake of iodine to reduce the risk for overcorrecting.

MS/MS-based newborn and family screening detects asymptomatic patients with very-long-chain acyl-CoA dehydrogenase deficiency.

OBJECTIVES: To determine whether asymptomatic persons with biochemical evidence of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency identified through expanded newborn screening with tandem mass spectometry have confirmed disease. STUDY DESIGN: We characterized 8 asymptomatic VLCAD-deficient individuals by enzyme and/or mutational analysis and compared them with clinically diagnosed, symptomatic patients with regard to mutations, enzyme activity, phenotype, and age of disease onset. RESULTS: VLCAD molecular analyses in 6 unrelated patients revealed the previously reported V243A mutation, associated with hepatic or myopathic phenotypes, on 7/12 alleles. All other mutations were also missense mutations. Residual VLCAD activities of 6% to 11% of normal were consistent with milder phenotypes. In these identified individuals treated prospectively with dietary modification as preventive measures, clinical symptoms did not develop during follow-up. CONCLUSIONS: MS/MS-based newborn screening correctly identifies VLCAD-deficient individuals. Based on mutational and enzymatic findings, these infants probably are at risk of future disease. Because life-threatening metabolic derangement can occur even in otherwise mild phenotypes, we advocate universal newborn screening programs for VLCAD deficiency to detect affected patients and prevent development of metabolic crises. Longer-term follow-up is essential to define outcomes, the definite risk of future disease, and appropriate treatment recommendations.

Lung lipid composition in zinc-deficient rats.

There have been a limited number of studies investigating surfactant lipid changes in lung with trace elements. The present investigation was designed to examine the effect of moderate zinc deficiency on the lipid metabolism in rat lung. We also evaluated whether zinc deficiency, which is a wide-spread problem, could play a role in adult respiratory distress syndrome (ARDS). For that purpose, adult male Wistar rats were fed two diets differing in zinc concentration. The rats were divided into two groups. One group was fed a zinc-deficient diet containing 3 mg Zn/kg, and the other group received a zinc-adequate control diet with 30 mg Zn/kg according to AIN 93-M. After 2 mon of treatment, we observed that in the zinc-deficient group (i) total lipids, phospholipids, and cholesterol increased whereas TG decreased in whole lung; (ii) phospholipid (PC) concentration increased in lamellar bodies and alveolar macrophages and decreased in extracellular surfactant but did not change in microsomes; (iii) protein concentration decreased in whole lung, extracellular surfactant, lamellar bodies, and macrophages; (iv) the incorporation of [Me-14C]choline into PC (phospholipids) of lung slices increased; and (v) the activity of CTP/phosphocholine cytidylyltransferase bound to the microsomes increased in the lung. These results suggest that the lipid concentration in the lung (especially the phospholipids) is modified directly or indirectly by a zinc-deficient diet. In a zinc-deficient diet, the lung changes the pattern of PC for an adaptive or recovery stage. Therefore, zinc deficiency implications are important for the design of therapies and public health interventions involving targeted zinc supplementation for high-risk groups or groups with certain diseases, such as ARDS.

Functional complementation of a genetic deficiency with human artificial chromosomes.

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology.

Micronutrient status and glutathione peroxidase in bedridden patients on tube feeding.

Deficiency of micronutrients, especially selenium, is common in critically ill patients. We investigated the micronutrient status (selenium, zinc, copper and manganese) and glutathione peroxidase (GSH-Px) activity in 30 tube-fed patients and 21 hospitalized non-tube-fed control patients. Serum levels of selenium, copper and manganese in tube-fed patients were significantly lower than in control patients (selenium: 4.85+/-1.38 microg/dl versus 8.67+/-1.45 pg/dl; copper: 44.7+/-36.9 microg/dl versus 92.1+/-21.2 microg/dl; manganese 0.59+/-0.41 microg/dl versus 1.52+/-0.59 microg/dl). However, zinc and log GSH-Px in the serum were similar in the two groups. Serum selenium concentration correlated with the daily intake of selenium in tube-fed patients, but zinc, copper and manganese concentrations did not correlate with the daily intake of the respective trace elements in tube-fed patients. Blood GSH-Px activity correlated positively with serum selenium concentrations in the control patients, but not in tube-fed patients. These results demonstrate that selenium content of enteral feed appears to be insufficient to maintain normal serum levels in elderly bedridden patients. Our findings emphasize the importance of monitoring micronutrient status in patients on enteral feeding to avoid trace element deficiencies.

A novel frameshift mutation in exon 23 of ATP7A (MNK) results in occipital horn syndrome and not in Menkes disease.

Menkes disease and occipital horn syndrome (OHS) are allelic, X-linked recessive copper-deficiency disorders resulting from mutations in ATP7A, or MNK. Classic Menkes disease has a severe phenotype, with death in early childhood, whereas OHS has a milder phenotype, with, mainly, connective-tissue abnormalities. Data suggest that steady-state localization of ATP7A to the trans-Golgi network (TGN) is necessary for proper activity of lysyl oxidase, which is the predominant cuproenzyme whose activity is deficient in OHS and which is essential for maintenance of connective-tissue integrity. Recently, it was reported that ATP7A-transcript levels as low as 2%-5% of normal are sufficient to result in the milder phenotype, OHS, rather than the phenotype of Menkes disease. In contrast to previously reported cases of OHS, we describe a case of OHS in which, because of a frameshift mutation, no normal ATP7A is produced. Although abundant levels of mutant transcript are present, there are substantially reduced levels of the truncated protein, which lacks the key dileucine motif L1487L1488. It has been demonstrated that the dileucine motif L1487L1488 functions as an endocytic signal for ATP7A cycling between the TGN and the plasma membrane. The present report is the first to describe an ATP7A truncation that results in OHS rather than in Menkes disease. The data from the present report support the concepts that (1) OHS results from lower levels of functional ATP7A and (2) ATP7A does not require the dileucine motif to function in copper efflux.

Iodine deficiency in cattle: compensatory changes in thyroidal selenoenzymes.

The trace elements selenium and iodine are both essential for normal thyroid hormone metabolism. To investigate the relationships between these functions, heifers were maintained on iodine-deficient or iodine-sufficient diets from mid pregnancy to term. In these heifers and their offspring the interrelationship between iodine and selenium was apparent with the preferential 10- to 12-fold induction of the selenoenzyme, thyroidal type I, selenium-containing iodothyronine deiodinase activity by iodine deficiency. This was accompanied by two- to four-fold increases in cytosolic glutathione peroxidase activity, probably reflecting increased oxidative activity and metabolism in the thyroid gland in response to iodine deficiency. The above selenoenzyme activities were not affected in liver, kidney, pituitary and brain by iodine deficiency. The results are consistent with a critical role for selenium in both the normal function of cattle thyroid and key enzymes to compensate for the effects of iodine deficiency.

Influence of selenium deficiency on vital functions in rats.

To clarify the relationship between selenium (Se) deficiency and functional disorders, the authors determined the Se concentration, anti-oxidant enzyme activity, and other parameters in rats fed a Se-deficient diet. Rats fed the Se-deficient diet showed a decrease in Se concentration and glutathione peroxidase (GSH-Px) activity in plasma, erythrocytes, heart, liver, and skeletal muscle from the first week after the initiation of the diet, an increase in heart lipid peroxide concentration from the second week, and an increase in liver glutathione S-transferase activity from the fourth week. From the twelfth week, a decrease in the growth rate in the rats fed the Se-deficient diet was observed. In spite of this growth impairment, no changes in electrocardiogram, muscle tone, degree of hemolysis, plasma biochemistry, or hematological values were detected. In summary, the authors found that a reduction of body Se is easily induced, but that the appearance of functional disorders following Se deficiency is difficult to detect in rats.