Lipid, Oxidative and Inflammatory Profile and Alterations in the Enzymes Paraoxonase and Butyrylcholinesterase in Plasma of Patients with Homocystinuria Due CBS Deficiency: The Vitamin B12 and Folic Acid Importance.

Cystathionine-ß-synthase (CBS) deficiency is the main cause of homocystinuria. Homocysteine (Hcy), methionine, and other metabolites of Hcy accumulate in the body of affected patients. Despite the fact that thromboembolism represents the major cause of morbidity in CBS-deficient patients, the mechanisms of cardiovascular alterations found in homocystinuria remain unclear. In this work, we evaluated the lipid and inflammatory profile, oxidative protein damage, and the activities of the enzymes paraoxonase (PON1) and butyrylcholinesterase (BuChE) in plasma of CBS-deficient patients at diagnosis and during the treatment (protein-restricted diet supplemented with pyridoxine, folic acid, betaine, and vitamin B12). We also investigated the effect of folic acid and vitamin B12 on these parameters. We found a significant decrease in HDL cholesterol and apolipoprotein A1 (ApoA-1) levels, as well as in PON1 activity in both untreated and treated CBS-deficient patients when compared to controls. BuChE activity and IL-6 levels were significantly increased in not treated patients. Furthermore, significant positive correlations between PON1 activity and sulphydryl groups and between IL-6 levels and carbonyl content were verified. Moreover, vitamin B12 was positively correlated with PON1 and ApoA-1 levels, while folic acid was inversely correlated with total Hcy concentration, demonstrating the importance of this treatment. Our results also demonstrated that CBS-deficient patients presented important alterations in biochemical parameters, possibly caused by the metabolites of Hcy, as well as by oxidative stress, and that the adequate adherence to the treatment is essential to revert or prevent these alterations.

Neurological damage in MSUD: the role of oxidative stress.

Maple syrup urine disease (MSUD) is a metabolic disease caused by a deficiency in the branched-chain α-keto acid dehydrogenase complex, leading to the accumulation of branched-chain keto acids and their corresponding branched-chain amino acids (BCAA) in patients. Treatment involves protein-restricted diet and the supplementation with a specific formula containing essential amino acids (except BCAA) and micronutrients, in order to avoid the appearance of neurological symptoms. Although the accumulation of toxic metabolites is associated to appearance of symptoms, the mechanisms underlying the brain damage in MSUD remain unclear, and new evidence has emerged indicating that oxidative stress contributes to this damage. In this context, this review addresses some of the recent findings obtained from cells lines, animal studies, and from patients indicating that oxidative stress is an important determinant of the pathophysiology of MSUD. Recent works have shown that the metabolites accumulated in the disease induce morphological alterations in C6 glioma cells through nitrogen reactive species generation. In addition, several works demonstrated that the levels of important antioxidants decrease in animal models and also in MSUD patients (what have been attributed to protein-restricted diets). Also, markers of lipid, protein, and DNA oxidative damage have been reported in MSUD, probably secondary to the high production of free radicals. Considering these findings, it is well-established that oxidative stress contributes to brain damage in MSUD, and this review offers new perspectives for the prevention of the neurological damage in MSUD, which may include the use of appropriate antioxidants as a novel adjuvant therapy for patients.

Experimental evidence for protein oxidative damage and altered antioxidant defense in patients with medium-chain acyl-CoA dehydrogenase deficiency.

The objective of this study was to test whether macromolecule oxidative damage and altered enzymatic antioxidative defenses occur in patients with medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency. We performed a cross-sectional observational study of in vivo parameters of lipid and protein oxidative damage and antioxidant defenses in asymptomatic, nonstressed, MCAD-deficient patients and healthy controls. Patients were subdivided into three groups based on therapy: patients without prescribed supplementation, patients with carnitine supplementation, and patients with carnitine plus riboflavin supplementation. Compared with healthy controls, nonsupplemented MCAD-deficient patients and patients receiving carnitine supplementation displayed decreased plasma sulfhydryl content (indicating protein oxidative damage). Increased erythrocyte superoxide dismutase (SOD) activity in patients receiving carnitine supplementation probably reflects a compensatory mechanism for scavenging reactive species formation. The combination of carnitine plus riboflavin was not associated with oxidative damage. These are the first indications that MCAD-deficient patients experience protein oxidative damage and that combined supplementation of carnitine and riboflavin may prevent these biochemical alterations. Results suggest involvement of free radicals in the pathophysiology of MCAD deficiency. The underlying mechanisms behind the increased SOD activity upon carnitine supplementation need to be determined. Further studies are necessary to determine the clinical relevance of oxidative stress, including the possibility of antioxidant therapy.

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy.

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n=14) and healthy age-matched controls (n=14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.

The effect of bone marrow transplantation on oxidative stress in X-linked adrenoleukodystrophy.

Oxidative stress plays an important role in the pathophysiology of neurodegenerative diseases, including X-linked adrenoleukodystrophy (X-ALD). In the present work, we evaluated lipid (malondialdehyde [MDA] content) and protein (sulfhydryl and carbonyl contents) oxidative damage parameters in plasma from X-ALD patients before and after bone marrow transplant (BMT), in order to verify if this treatment is capable to alter the oxidative parameters studied. We also evaluated the plasma concentration of hexacosanoic acid (C26:0) from X-ALD patients and correlated it with the oxidative damage parameters investigated. We observed that MDA content was significantly increased in plasma of X-ALD patients before BMT and after BMT when compared to controls, and that it was significantly reduced in plasma of X-ALD after BMT when compared to the before BMT group. These results indicate that lipid peroxidation is stimulated in X-ALD patients but there is a significant reduction of lipid peroxidation after BMT. Next, we observed a significant reduction of sulfhydryl content in plasma of X-ALD patients before BMT compared to controls indicating protein oxidative damage and that this measurement was increased in these patients after BMT as compared to before BMT. We found no significant differences in plasma carbonyl content in X-ALD patients before and after BMT as compared to controls. However, we observed a significant reduction in this parameter in X-ALD patients after BMT compared to before BMT. Finally, C26:0 plasma concentration was significantly reduced in X-ALD patients after BMT when compared to before BMT. We found no significant correlations between MDA and carbonyl values with C26:0 levels of the patients before BMT and after BMT, but a significant inverse correlation between sulfhydryl content and C26:0 levels was detected. In conclusion, the present study reinforces the hypothesis that lipid peroxidation and protein damage are induced in plasma of X-ALD patients and, in addition, demonstrates that BMT treatment is capable to reduce this pathogenic process. Taken together, the data obtained from plasma of X-ALD patients before and after BMT showing induction and protection, respectively, of oxidative stress, allowed to suggest that BMT, when well succeeded and under the recommendations, is effective to reduce C26:0 plasma levels and the increased lipid and protein oxidative damage in X-ALD.

Oxidative stress parameters in urine from patients with disorders of propionate metabolism: a beneficial effect of L:-carnitine supplementation.

Propionic (PA) and methylmalonic (MMA) acidurias are inherited disorders caused by deficiency of propionyl-CoA carboxylase and methylmalonyl-CoA mutase, respectively. Affected patients present acute metabolic crises in the neonatal period and long-term neurological deficits. Treatments of these diseases include a protein restricted diet and L: -carnitine supplementation. L: -Carnitine is widely used in the therapy of these diseases to prevent secondary L: -carnitine deficiency and promote detoxification, and several recent in vitro and in vivo studies have reported antioxidant and antiperoxidative effects of this compound. In this study, we evaluated the oxidative stress parameters, isoprostane and di-tyrosine levels, and the antioxidant capacity, in urine from patients with PA and MMA at the diagnosis, and during treatment with L: -carnitine and protein-restricted diet. We verified a significant increase of isoprostanes and di-tyrosine, as well as a significant reduction of the antioxidant capacity in urine from these patients at diagnosis, as compared to controls. Furthermore, treated patients presented a marked reduction of isoprostanes and di-tyrosine levels in relation to untreated patients. In addition, patients with higher levels of protein and lipid oxidative damage, determined by di-tyrosine and isoprostanes levels, also presented lower urinary concentrations of total and free L: -carnitine. In conclusion, the present results indicate that treatment with low protein diet and L: -carnitine significantly reduces urinary biomarkers of protein and lipid oxidative damage in patients with disorders of propionate metabolism and that L: -carnitine supplementation may be specially involved in this protection.

Oxidative stress in phenylketonuria: what is the evidence?

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by severe deficiency of phenylalanine hydroxylase activity, leading to the accumulation of phenylalanine and its metabolites in blood and tissues of affected patients. Phenylketonuric patients present as the major clinical feature mental retardation, whose pathomechanisms are poorly understood. In recent years, mounting evidence has emerged indicating that oxidative stress is possibly involved in the pathology of PKU. This article addresses some of the recent developments obtained from animal studies and from phenylketonuric patients indicating that oxidative stress may represent an important element in the pathophysiology of PKU. Several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in plasma and erythrocytes of PKU patients, which may be due to an increased free radical generation or secondary to the deprivation of micronutrients which are essential for these defenses. Indeed, markers of lipid, protein, and DNA oxidative damage have been reported in PKU patients, implying that reactive species production is increased in this disorder. A considerable set of data from in vitro and in vivo animal studies have shown that phenylalanine and/or its metabolites elicit reactive species in brain rodent. These findings point to a disruption of pro-oxidant/antioxidant balance in PKU. Considering that the brain is particularly vulnerable to oxidative attack, it is presumed that the administration of appropriate antioxidants as adjuvant agents, in addition to the usual treatment based on restricted diets or supplementation of tetrahydrobiopterin, may represent another step in the prevention of the neurological damage in PKU.

Prevention by L-carnitine of DNA damage induced by propionic and L-methylmalonic acids in human peripheral leukocytes in vitro.

Propionic acidemia (PAemia) and methylmalonic acidemia (MMAemia) are inborn errors of propionate metabolism characterized by the accumulation of, respectively, propionic and l-methylmalonic acids (and their metabolites) in the blood and tissues of affected patients. The conditions lead to severe metabolic complications in the neonatal period and to long-term neurological manifestations. Treatment for these disorders consists of a protein-restricted diet, supplemented with synthetic formulas of amino acids, but excluding isoleucine, threonine, valine and methionine; and l-carnitine, to promote detoxication. In vitro and in vivo studies have demonstrated that lipid and protein oxidative damage may be involved in the pathophysiology of these diseases, but DNA damage has not been fully investigated. In this work, we evaluated in vitro the effects of PA and MMA, in the presence or absence of l-carnitine, on DNA damage in peripheral leukocytes, as determined by the alkaline comet assay, using silver staining and visual scoring. PA and MMA induced a DNA damage index (DI) significantly higher than that of the control group. l-Carnitine significantly reduced PA- and MMA-induced DNA damage, in a concentration-dependent manner. Our findings indicate that PA and MMA induce DNA damage and l-carnitine is able to prevent this damage.

Effects of insulin and clonazepam on DNA damage in diabetic rats submitted to the forced swimming test.

Diabetes mellitus (DM) is a chronic hyperglycemic state. DM may be associated with moderate cognitive deficits and neurophysiologic/structural changes in the brain (diabetic encephalopathy). Psychiatric manifestations seem to accompany this encephalopathy, since the prevalence of depression in diabetic patients is much higher than in the general population, and clonazepam is being used to treat this complication. The excessive production of oxygen free radicals that may occur in diabetes induces a variety of lesions in macromolecules, including DNA. In this work, we analyzed DNA damage in leukocytes from streptozotocin-induced diabetic rats submitted to the forced swimming test. The DNA damage index was significantly elevated (DI=61.00 ± 4.95) in the diabetic group compared to the control group (34.00 ± 1.26). Significant reductions of the damage index were observed in diabetic animals treated with insulin (45.00 ± 1.82), clonazepam (52.00 ± 1.22), or both agents (39.00 ± 5.83, not significantly different from control levels). Insulin plus clonazepam can protect against DNA damage in stressed diabetic rats.

Urinary biomarkers of oxidative stress and plasmatic inflammatory profile in phenylketonuric treated patients.

Oxidative stress has been proposed as an important pathophysiologic feature of various inborn errors of metabolism, including phenylketonuria (PKU). Considering that there are few studies relating oxidative stress and inflammation directly in PKU disease, the aim of this study was to evaluate and correlate oxidative damage to biomolecules, antioxidant defenses, pro-inflammatory cytokines, phenylalanine (Phe) and its metabolites (phenyllactic acid--PLA and phenylacetic acid--PAA) levels in urine and plasma from patients with PKU under dietary treatment. We observed a marked increase of isoprostanes, which is a lipid peroxidation biomarker, in urine from these treated patients. Next, we demonstrated that protein oxidative damage, measured by di-tyrosine formation, was significantly increased in urine from PKU treated patients and that decreased urinary antioxidant capacity was also observed. Our findings concerning to the inflammatory cytokines interleukin-6 and interleukin-1ß, both significantly increased in these patients, provide evidence that the pro-inflammatory state occurs. Besides, interleukin-1ß was positively correlated with isoprostanes. We observed a negative correlation between interleukin-6 and interleukin-10, an anti-inflammatory cytokine. Di-tyrosine was positively correlated with Phe, which indicates oxidative damage to proteins, as well as with PAA. These findings may suggest that the protein damage may be induced by Phe and its metabolite PAA in PKU. Our results indicate that pro-oxidant and pro-inflammatory states occur and are, in part, correlated and protein oxidation seems to be induced by Phe and PPA in PKU patients.

L-carnitine supplementation as a potential antioxidant therapy for inherited neurometabolic disorders.

In recent years increasing evidence has emerged suggesting that oxidative stress is involved in the pathophysiology of a number of inherited metabolic disorders. However the clinical use of classical antioxidants in these diseases has been poorly evaluated and so far no benefit has been demonstrated. l-Carnitine is an endogenous substance that acts as a carrier for fatty acids across the inner mitochondrial membrane necessary for subsequent beta-oxidation and ATP production. Besides its important role in the metabolism of lipids, l-carnitine is also a potent antioxidant (free radical scavenger) and thus may protect tissues from oxidative damage. This review addresses recent findings obtained from patients with some inherited neurometabolic diseases showing that l-carnitine may be involved in the reduction of oxidative damage observed in these disorders. For some of these diseases, reduced concentrations of l-carnitine may occur due to the combination of this compound to the accumulating toxic metabolites, especially organic acids, or as a result of protein restricted diets. Thus, l-carnitine supplementation may be useful not only to prevent tissue deficiency of this element, but also to avoid oxidative damage secondary to increased production of reactive species in these diseases. Considering the ability of l-carnitine to easily cross the blood-brain barrier, l-carnitine supplementation may also be beneficial in preventing neurological damage derived from oxidative injury. However further studies are required to better explore this potential.

Reduction of butyrylcholinesterase activity in plasma from patients with disorders of propionate metabolism is prevented by treatment with L-carnitine and protein restriction.

OBJECTIVE: We investigated the relationship between butyrylcholinesterase (BuChE) activity and lipid oxidative damage in patients with disorders of propionate metabolism, before and after treatment with protein restriction and L-carnitine. DESIGN AND METHODS: BuChE activity and malondialdehyde (MDA) were measured in plasma from eight untreated patients (at diagnosis) and from seven patients under treatment with protein restriction and L-carnitne supplementation (100mg/kg/day). RESULTS: We verified a significant reduction of butyrylcholinesterase activity, as well as an increased MDA formation in plasma from untreated patients. However, treated patients presented MDA and BuChE activity similar to controls. Furthermore, butyrylcholinesterase activity was negatively correlated with MDA concentrations in these patients. CONCLUSION: The results suggest that an increased free radicals formation may be involved in the decrease of butyrylcholinesterase activity, possibly contributing to the neurological damage of these disorders, and that treatment with L-carnitine and low-protein diet possibly is able to prevent this damage.

Oxidative stress in Niemann-Pick type C patients: a protective role of N-butyl-deoxynojirimycin therapy.

Niemann-Pick type C (NPC) is a rare neurodegenerative disorder biochemically characterized by the accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes of the affected patients. N-butyl-deoxynojirimycin is the only approved drug for patients with NPC disease. It inhibits glycosphingolipid synthesis, therefore reducing intracellular lipid storage. Although the mechanisms underlying the neurologic damage in the NPC disease are not yet well established, in vitro and in vivo studies suggest an involvement of reactive species in the pathophysiology of this disease. In this work we aimed to evaluate parameters of lipid and protein oxidation, measured by thiobarbituric acid-reactive species (TBA-RS) and protein carbonyl formation, respectively, as well as the enzymatic and non-enzymatic antioxidant defenses in plasma, erythrocytes and fibroblasts from NPC1 patients, at diagnosis and during treatment with N-butyl-deoxynojirimycin. We found a significant increase of TBA-RS in plasma and fibroblasts, as well as increased protein carbonyl formation and decreased total antioxidant status (TAS) in plasma of untreated NPC1 patients as compared to the control group. In addition, erythrocyte glutathione peroxidase (GSH-Px) activity was increased, whereas CAT and SOD activities were normal in these patients. We also observed that patients treated with N-butyl-deoxynojirimycin normalized plasma TBA-RS and TAS, as well as erythrocyte GSH-Px activity. Taken together, the present data indicate that oxidative stress is increased in patients with NPC1 disease and that treatment with N-butyl-deoxynojirimycin is able to confer protection against this pathological process.

Reduction of lipid and protein damage in patients with disorders of propionate metabolism under treatment: a possible protective role of L-carnitine supplementation.

Disorders of propionate metabolism are autosomal recessive diseases clinically characterized by acute metabolic crises in the neonatal period and long-term neurological deficits whose pathophysiology is not completely established. There are increasing evidences demonstrating antioxidant properties for L-carnitine, which is used in the treatment of propionic and methylmalonic acidemias to increase the excretion of organic acids accumulated in tissues and biological fluids of the affected patients. In this work we aimed to evaluate lipid (malondialdehyde content) and protein (carbonyl formation and sulfhydryl oxidation) oxidative damage in plasma from patients with propionic and methylmalonic acidemias at the moment of diagnosis and during treatment with L-carnitine. We also correlated the parameters of oxidative damage with plasma total, free and esterified L-carnitine levels. We found a significant increase of malondialdehyde and carbonyl groups, as well as a reduction of sulfhydryl groups in plasma of these patients at diagnosis compared to controls. Furthermore, patients under treatment presented a marked reduction of the content of protein carbonyl groups, similar to controls, and malondialdehyde content in relation to patients at diagnosis. In addition, plasma total and free L-carnitine concentrations were negatively correlated with malondialdehyde levels. Taken together, the present data indicate that treatment significantly reduces oxidative damage in patients affected by disorders of propionate metabolism and that l-carnitine supplementation may be involved in this protection.