Alessandro Wajner

Application of a comprehensive protocol for the identification of gaucher disease in Brazil

Gaucher disease (GD) is a sphingolipidosis caused by a genetic defect that leads to glucocerebrosidase (β‐glucosidase) deficiency. Between January 1982 and October 2003, 1,081 blood samples from patients suspected of having GD were referred for biochemical analysis. The activities of the enzymes β‐glucosidase (β‐glu) and chitotriosidase (CT) were measured in these samples. Among the 412 diagnosed cases of GD (38.1%), the great majority were GD type 1. The Brazilian regions with the greatest concentration of these patients were the Southeast, South, and Northeast. The mean age of patients at diagnosis was 19 years. The activity of β‐glu in patients with GD was, on average, 10.7% of that of normal individuals. CT was, on average, 269 times more elevated in this group of patients. Among the 669 cases with no confirmation of GD, there were patients with Niemann–Pick disease types A, B, or C (44 cases), possible heterozygotes for GD (59 cases), patients with other lysosomal storage diseases (LSDs) (19 cases) or with other inborn errors of metabolism (3 cases). In 508 cases, no metabolic disorder was found. This study shows that the biochemical protocol employed was effective for the detection of GD, a disease that is reasonably frequent in Brazil.

Detection of Mucopolysaccharidosis Type I Heterozygotes Based on the Biochemical Characteristics of Leukocyte α-L-Iduronidase

BACKGROUND In the present study, we biochemically characterized the enzyme alpha-L-iduronidase (IDUA) of leukocytes from normal individuals and from mucopolysaccharidosis I (MPS I) heterozygotes, and compared these characteristics to discriminate for inclusion into two different groups. METHODS We fluorimetrically measured IDUA activity in leukocytes using 4-methylumbelliferyl-alpha-L-iduronide as an artificial substrate. Optimum pH, Km, Vmax, and thermostability of the enzyme at 50 degrees C were determined. RESULTS Based on leukocyte IDUA activity, we divided the heterozygotes into two groups, one (group 1) with activity below that detected in controls, and the other with activity similar to that of normal individuals (group 2). The optimum pH for IDUA was 2.7 for normal individuals and 2.6-2.8 for heterozygotes. With respect to Km, there was a difference only between the value for normal IDUA (0.60 mM) and the value for group 2 (0.38 mM), while group 1 showed a statistically similar value (0.49 mM). The Vmax of the reaction was discriminated in the three groups in a highly effective manner. The IDUA of normal individuals had a higher Vmax (60.98 nmoL/h x mg protein) than the enzyme of group 1 heterozygotes (28.66 nmoL/h x mg protein) and the enzyme of group 2 (31.78 nmoL/h x mg protein). When the IDUA from the three groups was pre-incubated at 50 degrees C, we observed that the IDUA of both group 1 and group 2 was significantly more thermostable than the IDUA of normal individuals. CONCLUSIONS Determination of IDUA activity alone is not sufficient to discriminate between MPS I heterozygotes and normal individuals because a considerable overlap occurs between them. Our study showed that leukocyte IDUA from MPS I heterozygotes differed from the normal enzyme in terms of optimum pH, Km, and Vmax of the reaction and thermostability at 50 degrees C. These parameters provide a simple and reliable tool for the detection of carriers for MPS I.

Detection of mucopolysaccharidosis type I heterozygotes on the basis of the biochemical properties of plasma α-l-iduronidase

BACKGROUND Mucopolysaccharidosis type I (MPS I) is a disease caused by deficiency of the enzyme alpha-L-iduronidase (IDUA). Since no treatment is currently available for this disorder, the detection of heterozygotes is very important for genetic counseling and prenatal diagnosis. The objective of the present study was to characterize plasma IDUA from MPS I heterozygotes in an attempt to distinguish it from that of normal individuals. METHODS We determined the optimum pH, Km, Vmax and Calpha (Vmax/Km) of the reaction and the thermal stability of IDUA at 50 degrees C. RESULTS MPS I heterozygotes can be separated from normal individuals on the basis of Km, Calpha and thermal stability of the enzyme. CONCLUSIONS Taking into consideration the clinical status of the homozygous offspring, we were able to subdivide the MPS I heterozygotes into various subgroups (Hurler, Scheie or Hurler/Scheie compound), and verified that the Hurler subgroup had a lower optimum pH for IDUA activity than controls and other MPS I subgroups, and that all MPS I subgroups had higher Km and lower Calpha when compared to controls.

cis-4-Decenoic and decanoic acids impair mitochondrial energy, redox and Ca(2+) homeostasis and induce mitochondrial permeability transition pore opening in rat brain and liver: Possible implications for the pathogenesis of MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized by tissue accumulation of octanoic (OA), decanoic (DA) and cis-4-decenoic (cDA) acids, as well as by their carnitine by-products. Untreated patients present episodic encephalopathic crises and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the effects of OA, DA, cDA, octanoylcarnitine (OC) and decanoylcarnitine (DC) on critical mitochondrial functions in rat brain and liver. DA and cDA increased resting respiration and diminished ADP- and CCCP-stimulated respiration and complexes II-III and IV activities in both tissues. The data indicate that these compounds behave as uncouplers and metabolic inhibitors of oxidative phosphorylation. Noteworthy, metabolic inhibition was more evident in brain as compared to liver. DA and cDA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded brain and liver mitochondria. The reduction of Ca(2+) retention capacity was more pronounced in liver and totally prevented by cyclosporine A and ADP, as well as by ruthenium red, demonstrating the involvement of mitochondrial permeability transition (mPT) and Ca(2+). Furthermore, cDA induced lipid peroxidation in brain and liver mitochondria and increased hydrogen peroxide formation in brain, suggesting the participation of oxidative damage in cDA-induced alterations. Interestingly, OA, OC and DC did not alter the evaluated parameters, implying lower toxicity for these compounds. Our results suggest that DA and cDA, in contrast to OA and medium-chain acylcarnitines, disturb important mitochondrial functions in brain and liver by multiple mechanisms that are possibly involved in the neuropathology and liver alterations observed in MCAD deficiency.

α-Ketoadipic Acid and α-Aminoadipic Acid Cause Disturbance of Glutamatergic Neurotransmission and Induction of Oxidative Stress In Vitro in Brain of Adolescent Rats

Tissue accumulation of α-ketoadipic (KAA) and α-aminoadipic (AAA) acids is the biochemical hallmark of α-ketoadipic aciduria. This inborn error of metabolism is currently considered a biochemical phenotype with uncertain clinical significance. Considering that KAA and AAA are structurally similar to α-ketoglutarate and glutamate, respectively, we investigated the in vitro effects of these compounds on glutamatergic neurotransmission in the brain of adolescent rats. Bioenergetics and redox homeostasis were also investigated because they represent fundamental systems for brain development and functioning. We first observed that AAA significantly decreased glutamate uptake, whereas glutamate dehydrogenase activity was markedly inhibited by KAA in a competitive fashion. In addition, AAA and more markedly KAA induced generation of reactive oxygen and nitrogen species (increase of 2′,7′-dichloroflurescein (DCFH) oxidation and nitrite/nitrate levels), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione (GSH)) and aconitase activity. Furthermore, KAA-induced lipid peroxidation and GSH decrease were prevented by the antioxidants α-tocopherol, melatonin, and resveratrol, suggesting the involvement of reactive species in these effects. Noteworthy, the classical inhibitor of NMDA glutamate receptors MK-801 was not able to prevent KAA-induced and AAA-induced oxidative stress, determined by DCFH oxidation and GSH levels, making unlikely a secondary induction of oxidative stress through overstimulation of glutamate receptors. In contrast, KAA and AAA did not significantly change brain bioenergetic parameters. We speculate that disturbance of glutamatergic neurotransmission and redox homeostasis by KAA and AAA may play a role in those cases of α-ketoadipic aciduria that display neurological symptoms.

Mevalonolactone disrupts mitochondrial functions and induces permeability transition pore opening in rat brain mitochondria: Implications for the pathogenesis of mevalonic aciduria

&NA; Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (&Dgr;&PSgr;m), NAD(P)H content and the capacity to retain Ca2+ in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca2+‐loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca2+ in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML‐induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA. HighlightsMevalonic acid (MA) and mevalonolactne (ML) accumulate in mevalonic aciduria (MVA).Patients affected by this disorder present brain dysfunction.Disruption of important mitochondrial functions and MPT are provoked by ML in brain.These pathomechanisms may be involved in MVA pathogenesis.

Effect of CuCl2, NaCl and EDTA on the enzyme α-l-iduronidase in the plasma of normal individuals and heterozygotes for MPS I

BACKGROUND It has been previously demonstrated that the enzyme alpha-L-iduronidase (IDUA) of patients with MPS I shows a different biochemical behavior in each of the three clinical forms of these. In heterozygotes, its biochemical behavior has been recently established in leukocyte and plasma samples, demonstrating that it is possible to distinguish individuals heterozygous for MPS I within an unselected population. METHODS We evaluated the effect of copper chloride, EDTA and sodium chloride on the activity of the enzyme alpha-L-iduronidase in the plasma of normal individuals and of MPS I heterozygotes and observed the type of inhibition caused, the Ki, the apparent Km and the apparent Vmax for each inhibitor. RESULTS Sodium chloride inhibited the enzyme in normal individuals and in 40% of the heterozygotes evaluated and activated it in 60% of heterozygotes. The remaining compounds inhibited IDUA in both heterozygotes and normal individuals. CONCLUSIONS We detected significant differences capable of differentiating MPS I heterozygotes from normal individuals by simply adding sodium chloride, EDTA or copper chloride to the incubation medium at the time of IDUA activity determination, with a potential use in carrier detection protocols.

High vulnerability of the heart and liver to 3-hydroxypalmitic acid-induced disruption of mitochondrial functions in intact cell systems: CECATTO et al.

Patients affected by long‐chain 3‐hydroxyacyl‐CoA dehydrogenase (LCHAD) deficiency predominantly present severe liver and cardiac dysfunction, as well as neurological symptoms during metabolic crises, whose pathogenesis is still poorly known. In this study, we demonstrate for the first time that pathological concentrations of 3‐hydroxypalmitic acid (3HPA), the long‐chain hydroxyl fatty acid (LCHFA) that most accumulates in LCHAD deficiency, significantly decreased adenosine triphosphate‐linked and uncoupled mitochondrial respiration in intact cell systems consisting of heart fibers, cardiomyocytes, and hepatocytes, but less intense in diced forebrain. 3HPA also significantly reduced mitochondrial Ca2+ retention capacity and membrane potential in Ca2+‐loaded mitochondria more markedly in the heart and the liver, with mild or no effects in the brain, supporting a higher susceptibility of the heart and the liver to the toxic effects of this fatty acid. It is postulated that disruption of mitochondrial energy and Ca2+ homeostasis caused by the accumulation of LCHFA may contribute toward the severe cardiac and hepatic clinical manifestations observed in the affected patients.

Metabolite accumulation in VLCAD deficiency markedly disrupts mitochondrial bioenergetics and Ca2+ homeostasis in the heart

We studied the effects of the major long‐chain fatty acids accumulating in very long‐chain acyl‐CoA dehydrogenase (VLCAD) deficiency, namely cis‐5‐tetradecenoic acid (Cis‐5) and myristic acid (Myr), on important mitochondrial functions in isolated mitochondria from cardiac fibers and cardiomyocytes of juvenile rats. Cis‐5 and Myr at pathological concentrations markedly reduced mitochondrial membrane potential (ΔΨm), matrix NAD(P)H pool, Ca2+ retention capacity, ADP‐ (state 3) and carbonyl cyanide 3‐chlorophenyl hydrazine‐stimulated (uncoupled) respiration, and ATP generation. By contrast, these fatty acids increased resting (state 4) respiration (uncoupling effect) with the involvement of the adenine nucleotide translocator because carboxyatractyloside significantly attenuated the increased state 4 respiration provoked by Cis‐5 and Myr. Furthermore, the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP, as well as the Ca2+ uptake blocker ruthenium red, fully prevented the Cis‐5‐ and Myr‐induced decrease in ΔΨm in Ca2+‐loaded mitochondria, suggesting, respectively, the induction of MPT pore opening and the contribution of Ca2+ toward these effects. The findings of the present study indicate that the major long‐chain fatty acids that accumulate in VLCAD deficiency disrupt mitochondrial bioenergetics and Ca2+ homeostasis, acting as uncouplers and metabolic inhibitors of oxidative phosphorylation, as well as inducers of MPT pore opening, in the heart at pathological relevant concentrations. It is therefore presumed that a disturbance of bioenergetics and Ca2+ homeostasis may contribute to the cardiac manifestations observed in VLCAD deficiency.

Mechanistic Bases of Neurotoxicity Provoked by Fatty Acids Accumulating in MCAD and LCHAD Deficiencies

Fatty acid oxidation defects (FAODs) are inherited metabolic disorders caused by deficiency of specific enzyme activities or transport proteins involved in the mitochondrial catabolism of fatty aci...