Oxidative Stress in Homocystinuria Due to Cystathionine ß-Synthase Deficiency: Findings in Patients and in Animal Models

Abstract

Homocystinuria is an inborn error of amino acid metabolism caused by deficiency of cystathionine ß-synthase (CBS) activity, biochemically characterized by homocysteine (Hcy) and methionine (Met) accumulation in biological fluids and high urinary excretion of homocystine. Clinical manifestations include thinning and lengthening of long bones, osteoporosis, dislocation of the ocular lens, thromboembolism, and mental retardation. Although the pathophysiology of this disease is poorly known, the present review summarizes the available experimental findings obtained from patients and animal models indicating that oxidative stress may contribute to the pathogenesis of homocystinuria. In this scenario, several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in individuals affected by this disease. Furthermore, markers of lipid, protein, and DNA oxidative damage have been reported to be increased in blood, brain, liver, and skeletal muscle in animal models studied and in homocystinuric patients, probably as a result of increased free radical generation. On the other hand, in vitro and in vivo studies have shown that Hcy induces reactive species formation in brain, so that this major accumulating metabolite may underlie the oxidative damage observed in the animal model and human condition. Taken together, it may be presumed that the disruption of redox homeostasis may contribute to the tissue damage found in homocystinuria. Therefore, it is proposed that the use of appropriate antioxidants may represent a novel adjuvant therapy for patients affected by this disease.

Introduction

Homocystinuria is an autosomal recessive disorder of amino acid metabolism usually caused by deficiency of cystathionine ß-synthase (CBS), an enzyme that catalyzes the transsulfuration of homocysteine (Hcy) to cystathionine with the help of its cofactor pyridoxine (Fig. 1). Untreated patients accumulate Hcy and methionine (Met) in biological fluids and have a high urinary excretion of homocystine (homocysteine–cysteine complex), the oxidized product of Hcy. The prevalence of homocystinuria ranges from 1:58,000 to 1:1,000,000 newborns (Mudd et al. 2001).

Fig. 1.

Metabolic pathway of homocysteine metabolism and the blockage of cystathionine ß-synthase (homocystinuria), the enzyme which catalyzes cystathionine synthesis from homocysteine and serine. The metabolic blockage results in homocysteine and methionine accumulation, as well as cystine deficiency and high homocystine excretion. Defects of transmethylation (methionine → homocysteine), transsulfuration (methionine → sulfate), and remethylation (homocysteine → methionine) enzymes of sulfur amino acid metabolism: 10.1, methionine adenosyltransferase; 10.2, cystathionine β-synthase; a0.3, γ-cystathionase; 10.4, sulfite oxidase; 10.5, molybdenum cofactor; 10.6, methylenetetrahydrofolate reductase; 10.7 and 10.8, methionine synthase (Adapted from Skovby, 2003)

Besides deficient activity of CBS, other genetic defects, such as mutations inactivating the enzymes methionine synthase and methylenetetrahydrofolate reductase (MTHFR), may lead to increased concentrations of Hcy in plasma (Mudd et al. 2001). Acquired causes of moderately elevated plasma levels of Hcy include deficiencies of the cofactors vitamins B12, B6 and folate, increasing age, disorders such as cardiovascular diseases, neurological diseases, renal failure and hypothyroidism, drugs that interfere with the metabolism of the cofactors, and lifestyle factors, including cigarette smoking, alcoholism, diet, and physical inactivity (Skovierová et al. 2016).

CBS deficiency is clinically characterized by heterogeneous clinical manifestations in various organs and tissues, such as thinning and lengthening of the long bones, osteoporosis, dislocation of the ocular lens, thromboembolism, and mental retardation (Mudd et al. 2001). Thromboembolic complications occurring in arteries and veins are a major cause of morbidity and mortality. Prognosis depends on the location and extent of vascular occlusion, being thrombophlebitis and pulmonary embolism the most common vascular accidents found in patients with homocystinuria (Andria et al. 2006). Although intelligence may be normal in some cases, many individuals with homocystinuria present progressive mental retardation (IQ range from 10 to 138) (Yap et al. 2001). There are two different clinical forms of CBS deficiency: pyridoxine-responsive (B₆-responsive) and pyridoxine-non-responsive (B₆-non-responsive) (Mudd et al. 1985). B₆-responsive patients (mean IQ is 79) are more likely than individuals with the B₆-non-responsive homocystinuria (mean IQ is 57) to be cognitively intact or only mildly affected (Yap et al. 2001). Some CBS-deficient patients may also develop psychiatric disturbances, including personality disorder, anxiety, depression, obsessive–compulsive behavior, and psychotic episodes (Hidalgo Mazzei et al. 2014). Uncontrolled electrical activity in the brain (seizures) occurs in 21% of untreated patients (Hidalgo Mazzei et al. 2014).

Treatment for B6-responsive patients consists of vitamin B6 supplementation, at a dose of approximately 200 mg/day, and also protein-restricted diet. B6-non-responsive patients require restriction of Met intake and cystine supplementation (Rao et al. 2008). An alternate therapy can be the use of betaine, 6–9 g/day (Wilcken and Wilcken 1997). Moreover, folate and vitamin B12 are also used (5 mg/day of folic acid and 1 mg intramuscular injection per month of vitamin B12 in the form of hydroxocobalamin) (Rao et al. 2008).

Although the pathophysiology of homocystinuria is not yet well established, it was proposed that Hcy excess or cysteine deficiency rather than the accumulation of Met is more likely to cause the pathogenesis of this disease (Mudd et al. 2001). On the other hand, it has been suggested that oxidative stress may play an important role in the pathophysiology of homocystinuria (Lentz 1998; Wilcken et al. 2000; Vilaseca-Buscà et al. 2002; Faraci and Lentz 2004; Vanzin et al. 2011, 2014, 2015). This was based on studies carried out in animal models that found an association between Hcy and oxidative stress (Wyse et al. 2002; Streck et al. 2002a, b; Robert et al. 2005; Machado et al. 2011; da Cunha et al. 2011; Kolling et al. 2014).

Oxidative Stress, Free Radicals, and Antioxidant Defenses

Free radicals and other reactive species play an important role in the pathogenesis of various human diseases (Halliwell and Whiteman 2004). Free radicals are chemical structures with one or more unpaired electrons occupying a single molecular or atomic orbital, which gives a high reactivity and instability to the molecule. These molecules are capable of independent existence and can lead to cell damage (Halliwell and Gutteridge 2007). These radicals can be endogenously formed by different mechanisms in both physiological and pathological conditions, as well as by exogenous factors, such as drugs, air pollutants, radiation, and chemical agents (Halliwell 1991; Smith et al. 2005).

Antioxidant is any substance that inhibits substrate oxidation. Antioxidants can be classified according to their function as follows: free radical scavenger (e.g., ascorbic acid), scavenger of non-radical oxidants (e.g., catalase, thiols), and compounds that inhibit the generation of oxidants (e.g., metal chelators) or that induce the production of antioxidants (e.g., isothiocyanates). They can also be classified as endogenous or exogenous, pending on their origins. Exogenous antioxidants come from the diet or supplementation. Examples are vitamins E, C, carotenoids (present in fruits with red color), and carnitine (found in foods of animal origin). Endogenous antioxidants can be classified into enzymatic or non-enzymatic substances. Enzymatic defenses against oxidative damage include enzymes involved in neutralizing reactive species, such as superoxide dismutase, catalase, and glutathione reductase. Non-enzymatic antioxidants are substances produced by the organism, as albumin, transferrin, and ceruloplasmin with activity against pro-oxidant substances (Halliwell 1991; Halliwell and Gutteridge 2007).

Oxidative stress is defined as an imbalance between reactive species production and tissue antioxidant defense content that can be due to increased generation of reactive species and/or decreased levels of antioxidants. Increasing evidence has shown that tissue damage caused by reactive species is an important contributing factor in chronic inflammatory, vascular, neoplastic, and neurodegenerative diseases (Halliwell and Gutteridge 2007). Apart from the important role of oxidative stress in neurodegenerative diseases, various studies have shown that oxidative stress participates in the pathophysiology of various inborn errors of metabolism (IEM), including aminoacidopathies and organic acidemias (Vilaseca-Buscà et al. 2002; Wajner et al. 2004). Growing data indicate that the accumulation of toxic metabolites provokes an increase of free radicals and diminution of the antioxidant defenses in these diseases (Wajner et al. 2004). In the present work, we aim to present the available data in the literature indicating that oxidative stress occurs in homocystinuric patients and in animal models of this disease and may help understand the pathogenesis of homocystinuria.

Oxidative Stress in Homocystinuric Patients

Proteins, lipid, and DNA oxidative damage, as well as the antioxidant defenses and the proinflammatory profile have been investigated in homocystinuric patients (Table 1). In this regard, Vanzin and colleagues (2011, 2015) demonstrated that carbonyl content is increased in plasma of non-treated homocystinuric patients and partially reduced by treatment based on protein restriction with supplementation of pyridoxine, folate, betaine, and vitamin B12, when compared with healthy individuals, indicating that protein oxidative damage occurs in these patients and can be prevented by therapy. It was also observed that malondialdehyde (MDA) levels, an index of lipid peroxidation, were significantly higher in non-treated patients when compared to treated patients and healthy individuals. Furthermore, the treatment attenuated lipid oxidative damage in homocystinuric patients (Vanzin et al. 2011). It was also found that sulfhydryl content and the total antioxidant status (TAS), which indicates the quantity of tissue antioxidants, were significantly lower in homocystinuric patients at diagnosis and in patients under treatment (Vanzin et al. 2011, 2015), suggesting that the treatment was not able to prevent the decrease in the antioxidant defenses found at diagnosis. The same investigators found that the treated patients presented a significant decrease of both Hcy and Met when compared with patients at diagnosis, although these values remained above the normal range. Finally, it was found a significant negative correlation between sulfhydryl group content and Hcy levels, in contrast to a positive correlation between MDA levels and Hcy levels, suggesting a potential mechanistic role for Hcy in the oxidative damage observed in homocystinuria. Regarding Met, no correlation was found between this amino acid and the oxidative stress parameters, which reinforces the assumption that Met and its derivatives make little contribution to the oxidative damage in CBS deficiency (Vanzin et al. 2011, 2015). It was also verified that DNA damage was significantly higher in CBS-deficient patients when compared to healthy individuals (Vanzin et al. 2014). Since Hcy induces DNA damage in vivo and in vitro in white blood cells, it may be presumed that DNA damage in these patients can be correlated with the high plasma Hcy levels.

Table 1.

Oxidative stress and inflammatory parameters evaluated in biological fluids from homocystinuric patients at diagnosis and during dietary treatment

Parameter	Diagnosis	Treatment	Reference
Lipid peroxidation
MDA levels	Increased*	Increased	(Vanzin et al. 2011)
8-iso-prostaglandin F2α	NM	Increased	(Davi et al. 2001)
11-dehydrothromboxane	NM	Increased	(Davi et al. 2001)
Protein oxidation
Carbonyl content	Increased*	Increased	(Vanzin et al. 2011, 2015)
Sulfhydryl content	Decreased*	Decreased	(Vanzin et al. 2011, 2015)
DNA damage	NM	Increased	(Vanzin et al. 2014)
Antioxidant defense
TAS levels	Decreased*	Decreased	(Vanzin et al. 2011)
Vitamin B12	Decreased*	Decreased	(Vanzin et al. 2015)
Vitamin E	NM	Increased	(Davi et al. 2001)
Folic acid	Decreased*	Increased	(Vanzin et al. 2015)
SOD	NM	Increased	(Vilaseca-Buscà et al. 2002)
EC-SOD	Increased	Increased	(Wilcken et al. 2000)
Inflammatory profile
IL-6	Increased*	Increased	(Vanzin et al. 2015)
IFN-y	Similar*	Similar	(Vanzin et al. 2015)
IL-1β	Similar*	Similar	(Vanzin et al. 2015)

NM not measured

*at late diagnosis

Alterations of the lipid [total cholesterol, HDL cholesterol, LDL cholesterol, oxidized LDL cholesterol, apolipoprotein A1 (ApoA-1)] and the proinflammatory [interleukin-6 (IL-6), interleukin-1β (IL-1β), interferon-γ (IFN-γ)] profiles, as well as the activities of paraoxonase (PON1) and butyrylcholinesterase (BuChE), were also observed in the plasma of non-treated and treated CBS-deficient patients (Vanzin et al. et al. 2015). It was also demonstrated a correlation between some of these parameters with Hcy, folic acid, and vitamin B12 concentrations. Regarding the lipid profile, it was found a significant decrease in total cholesterol, HDL, and ApoA-1 levels in homocystinuric patients. Furthermore, PON1 activity was decreased in non-treated and treated homocystinuric patients, whereas BuChE activity was increased only in the untreated patients. Significant positive correlations between PON1 activity and sulfhydryl content and between HDL and ApoA-1 levels were also observed. Besides, it was demonstrated that IL-6 was significantly higher in non-treated patients and a moderate reduction of these levels in patients under treatment. A significant positive correlation between IL-6 levels and carbonyl group content, suggesting an association between inflammation and oxidative protein damage, was also observed, as well as a significant positive correlation between vitamin B12 and ApoA-1 levels, and between vitamin B12 levels and PON1. These findings suggest that vitamin B12 could be essential to increase ApoA-1 levels and PON1 activity in CBS-deficient patients. Additionally, it was demonstrated a significant negative correlation between folic acid and total Hcy concentrations. Folic acid was also increased in treated patients when compared to non-treated patients (Vanzin et al. 2015). Another study carried out by Pullin et al. (2002) verified that vitamin C therapy ameliorates the endothelial dysfunction in patients with homocystinuria, independent of changes in Hcy concentration. The authors concluded that vitamin C should be considered as an additional adjunct to therapy to reduce the potential long-term risk of atherothrombotic disease. Altogether, these data may be related to the important atheroprotective effects of these vitamins (Liao et al. 2006; Mikael et al. 2006; Devlin and Lentz 2006) that could, at least in part, decrease or revert the vascular alterations found in these patients.

A significant positive correlation was also observed between plasma Hcy levels and urinary 8-iso-prostaglandin F2α in CBS-deficient patients (Davi et al. 2001). In this context, it is emphasized that F2-isoprostanes represent a family of bioactive prostaglandin F2-like compounds that are produced from arachidonic acid through a non-enzymatic process of lipid peroxidation. Interestingly, 8-iso-prostaglandin F2α is an F2-isoprostane that induces vasoconstriction and modulates the function of human platelets, apart from being associated with several cardiovascular risk factors. It was also verified that urinary 8-iso-prostaglandin F2α and 11-dehydrothromboxane B2 excretion was significantly higher in CBS-deficient patients, whereas vitamin E supplementation was able to significantly decrease the urinary excretion of these substances. These preliminary observations suggest a potential role for antioxidant therapy attenuating Hcy-dependent oxidative changes that may promote atherothrombosis in this disease.

On the other hand, an important component of the endogenous antioxidant defense opposing the deleterious vascular effects of free radicals is superoxide dismutase (SOD), present in the vascular wall. Among the existing SOD isoenzymes, more of 90% of interstitial SOD is extracellular superoxide dismutase (EC-SOD). In this regard, it was demonstrated a significant positive correlation between EC-SOD and total Hcy in CBS-deficient patients, what could represent a protective antioxidant response to Hcy-induced oxidative damage and contribute to reducing cardiovascular risk in homocystinuric patients (Wilcken et al. 2000).

Finally, Vilaseca-Buscà et al. (2002) evaluated the antioxidant status in patients affected by inborn errors of metabolism (including CBS-deficient patients) through the measurement of erythrocyte antioxidant enzyme activities and demonstrated that SOD activity was significantly higher in these patients, possibly corroborating with the previous study.

Animal Models of Homocystinuria: Oxidative Stress Findings

Animal models are useful to better understand the pathophysiology of human diseases. In this context, a previous study investigated the effects of chronic hyperhomocysteinemia (Hhcy) on some parameters of oxidative damage, including catalase (CAT), SOD, glutathione peroxidase (GPx), total radical-trapping antioxidant potential (TRAP), and DNA damage in blood and parietal cortex of rats (Matté et al. 2009a). In order to evaluate whether DNA damage was permanent after cell division, it was also investigated the effect of Hcy on the micronucleus test, and it was shown that Hcy did not alter micronucleus frequency in blood of rats. It was also evaluated the effect of folic acid on the biochemical alterations elicited by Hhcy. It was shown that Hcy administration increased DNA damage and reduced TRAP, CAT, and GPx activities in parietal cortex. Furthermore, hyperhomocysteinemia provoked an increase of SOD and CAT activities, whereas TRAP was decreased and GPx activity was not altered in blood of rats. It was also verified that folic acid concurrent administration per se did not alter the activities of CAT, SOD, GPx, TRAP, and DNA damage but prevented the inhibition of these parameters caused by Hcy. The results confirmed that the supplementation with folic acid can be used as an adjuvant therapy in disorders that accumulate Hcy, as an antioxidant and for DNA stability.

Another study revealed that chronic administration of Hcy significantly reduced the antioxidant CAT and SOD enzyme activities, as well as glutamate uptake and Na(+),K(+)-ATPase activity, an enzyme highly vulnerable to oxidative damage, in the hippocampus of rats. The same treatment increased 2′,7′-dichlorofluorescein (DCFH) oxidation. Interestingly, these deleterious effects were prevented by vitamin C administration implying the involvement of reactive species (Machado et al. 2011).

Furthermore, it was evaluated the effect of acute Hcy administration on some parameters of oxidative stress, TRAP, CAT, SOD, and GPx and on Na(+),K(+)-ATPase activity and the effect of chronic pretreatment with vitamins E and C in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na(+),K(+)-ATPase, and CAT activities, without affecting the activities of SOD and GPx. The chronic pretreatment with vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na(+),K(+)-ATPase, and CAT activities caused by Hcy (Wyse et al. 2002).

Other investigators demonstrated that acute administration of Hcy significantly decreases memory acquisition, consolidation, and retrieval that were prevented by simultaneous administration of vitamins E and C, suggesting that memory dysfunction could be secondary to oxidative stress (Reis et al. 2002).

On the other hand, in vitro studies revealed that the addition of Hcy to the medium increased thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, and decreased TRAP (total non-enzymatic antioxidants), but did not change the antioxidant enzyme activities SOD, CAT, and GPx in rat hippocampus (Streck et al. 2003), thereby reinforcing the disruption of redox homeostasis provoked by Hcy in vivo.

Other work carried out in rat liver showed that chronic Hcy administration provoked a reduction of TRAP and total antioxidant reactivity (TAR), as well as total thiol and sulfhydryl group content, reduced glutathione concentrations, and CAT activity, indicating a severe impairment of liver antioxidant defenses (Matté et al. 2009b). Furthermore, lipid peroxidation, assessed by chemiluminescence and TBARS assay, was increased by Hcy. Furthermore, histological analysis in animals submitted to the same treatment induced inflammatory infiltration, fibrosis, and reduced content of glycogen/glycoprotein in liver sections. Despite the fact that several studies in experimental models of hyperhomocysteinemia in rats provide evidence for increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, associated with reactive oxygen species (ROS) production and hepatic lipid peroxidation, Matté et al. (2009b) did not observe any significant change in aminotransferases activities in tissue tested which they attributed to the short period of treatment with Hcy. These authors suggested that the liver biochemical and histological data elicited by Hcy could contribute to explain, at least in part, the mechanisms involved in hepatic damage associated with Hhcy.

Histologic evaluation of liver in CBS-deficient mice showed inflammation, fibrosis, and hepatic steatosis concomitant with an enhanced expression of tissue inhibitor of metalloproteinase-1, α-smooth muscle actin, pro(α)1 collagen type I, transforming growth factor-β1, and proinflammatory cytokines. The formation of carbonyl groups and the levels of MDA and 4-hydroxyalkenal (4-HNE) were also increased in the liver of the mice, implying enhanced protein and lipid oxidation. Moreover, the absence of caspase-3 activation, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL)-positive cells showed that protective signals may counteract apoptotic signals in the liver of CBS-deficient mice (Robert et al. 2005).

In another study using a genetic model of homocystinuria to avoid Hcy-independent side effects induced by dietary modifications, male CBS ± mice (intermediate model) and CBS +/+ males from the same litter aged 6 to 10 months were used to study the mechanisms of hepatic Hcy toxicity (Hamelet et al. 2008). The results showed that there was no accumulation of ROS (superoxide anion) and reactive nitrogen species (RNS) in the liver of CBS-deficient mice, although NADPH oxidase was activated. This ability of the liver to maintain its redox status could be due to a significant increase in CAT, whose activity was increased but not due to its overexpression. Since CAT activity is regulated by modulation of its phosphorylation, activation of the protein kinase A (PKA) by Hcy in hepatocytes may explain the observed increase in CAT activity. It was also found that GPx, glutathione reductase (GRed), and glutathione S-transferase (GST) activities in the liver of CBS ± mice were similar to those observed in CBS +/+ mice, indicating that GSH-dependent detoxification of hydrogen peroxide (H₂O₂) or oxidized lipids was not caused by Hcy. There was also no activation of SOD in the liver of CBS ± mice and a 25% decrease in hepatic H₂O₂ concentration in CBS ± mice compared to their CBS +/+ aggregates was observed.

Zhang et al. (2012) examined the causative role and mechanism of Hhcy in atherogenesis and monocyte differentiation using a novel mouse model of Hhcy and hyperlipidemia, in which CBS and low-density lipoprotein receptor (LDLr) genes were deficient (LDLr −/− CBS∓). Severe Hhcy was induced in this model by a high methionine diet containing adequate levels of B vitamins. Plasma Hcy levels were lowered from 244 µmol/L to 46 µmol/L by vitamin supplementation, which elevated the plasma folate levels. Hhcy accelerated atherosclerosis and promoted inflammatory monocyte differentiation in both bone marrow and tissue origins in the aortas and peripheral tissues. Plasma levels of TNF-α, IL-6, and monocyte chemotactic protein-1 (MCP-1) were elevated, as well as vessel wall monocyte accumulation and increased macrophage maturation. Hcy-lowering therapy reversed Hhcy-induced lesion formation, plasma cytokine increase, and blood and vessel inflammatory monocyte accumulation probably due to elevation of plasma folate levels. It was also observed that plasma Hcy levels were positively correlated with the plasma levels of proinflammatory cytokines and that Hhcy induced inflammatory monocyte differentiation leading to proinflammatory cytokine production and systemic inflammation and promoted inflammatory monocyte subset differentiation via oxidative stress in primary splenocytes.

It was also investigated whether creatine could prevent the induced oxidative stress provoked by chronic Hhcy in skeletal muscle of rats (Kolling et al. 2014). It was first shown that chronic Hcy administration increased the DCF oxidation, an index of production of reactive species, and the TBARS levels, an index of lipid peroxidation. Antioxidant enzyme, SOD, and CAT activities were also increased, but GPx activity was not altered. The contents of glutathione (GSH), sulfhydryl, and carbonyl were decreased, as well as nitrite levels. Moreover, concurrent administration of creatine prevented some Hcy effects probably by its antioxidant properties. Based on these findings, it was suggested that creatine may be used as an adjuvant therapy for ameliorating the symptoms associated with oxidative insult that are found in homocystinuric patients.

Another study performed in rat lung revealed that chronic Hhcy significantly increased TBARS and protein carbonyl content, as well as reactive species generation evaluated by DCF fluorescence assay, suggesting that this amino acid also causes lipid and protein oxidative damage through free radicals in this tissue (Cunha et al. 2011). CAT, GPx, and glucose 6-phosphate dehydrogenase (G6PD) activities, as well as TRAP and GSH concentrations, were significantly reduced by Hhcy, suggesting that this amino acid causes a reduction of both enzymatic and non-enzymatic antioxidants. Since nitrate and nitrite levels were not changed by this treatment, it is presumed that ROS, rather than RNS, was involved in these alterations.

On the other hand, chronic administration of Hcy selectively decreased the activity of synaptosomal Na(+),K(+)-ATPase, an enzyme very susceptible to oxidative attack, without altering Mg(2 +)-ATPase activity in the hippocampus of treated rats (Streck et al. 2002a). These in vivo effects were corroborated by other findings showing that Hcy, at concentrations usually found in homocystinuria, but not Met that participates in Hcy metabolism, inhibits Na(+),K(+)-ATPase activity in the hippocampus in vitro (Streck et al. 2001). It was also demonstrated that the antioxidant Trolox (vitamin E analogue) prevented the inhibitory effect of Hcy on Na(+),K(+)-ATPase activity (Streck et al. 2001). Besides, Streck et al. (2002b) determined the in vitro effects of Hcy and Met on Na(+),K(+)-ATPase and Mg(2 +)-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na(+),K(+)-ATPase but not Mg(2 +)-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, the simultaneous addition of cysteine to the medium with hippocampal homogenates and Hcy prevented the decrease of Na(+),K(+)-ATPase activity induced by Hcy. These findings indicate that the oxidation of critical groups in the enzyme may possibly be involved in Hcy inhibitory effect. Furthermore, since Na(+),K(+)-ATPase is a fundamental enzyme responsible for maintaining the ionic gradient necessary for neuronal excitability and it is present at high concentrations in the brain cellular membrane (Erecinska and Silver 1994), and, on the other, the decrease of this activity is found in various neurodegenerative disorders (Lees 1993; Streck et al. 2002a), it is presumed that the inhibition of this enzyme activity by Hcy possibly contributes to the neurological dysfunction found in homocystinuric patients.

Finally, it was proposed that the measurement of Na(+),K(+)-ATPase and BuChE activities in platelets and plasma of rats, respectively, may represent useful peripheral markers for the neurotoxic effects of Hcy since these activities were significantly inhibited in vitro and in vivo by Hcy (Schulpis et al. 2006; Stefanello et al. 2003, 2005). Interestingly, the reduction of BuChE activity caused by acute administration of Hcy was prevented in rats pretreated for one week with vitamins E and C, suggesting that this enzyme protein suffered oxidative attack. It is of note that BuChE is present in all tissues, including serum, heart, vascular endothelia, and nervous system (Prody et al. 1987).

Table 2 describes the oxidative stress parameters studied in tissues and biological fluids from the animal models of homocystinuria. These findings show a consistent profile of oxidative stress in the brain, blood, liver, muscle, and lung of rats provoked by Hcy. It is therefore presumed that the disruption of redox homeostasis may represent an important mechanism contributing to the clinical phenotype occurring in homocystinuric patients.

Table 2.

Oxidative stress parameters evaluated in tissues and biological fluids from animal models of homocystinuria

Parameter	In vitro/acute/chronic	Reference
Lipid peroxidation	Increased	(Streck et al. 2003; Robert et al. 2005; da Cunha et al. 2011; Kolling et al. 2014; Matté et al. 2009b)
Protein oxidation	Increased	(Robert et al. 2005; da Cunha et al. 2011)
DNA damage	Increased	(Matté et al. 2009a)
Antioxidant defense	Decreased	(Wyse et al. 2002; Streck et al. 2003; Matté et al. 2009a, b; da Cunha et al. 2011)
Antioxidant enzyme	Altered	(Wyse et al. 2002; Streck et al. 2003; Matté et al. 2009a, b; Machado et al. 2011; da Cunha et al. 2011; Hamelet et al. 2008; Kolling et al. 2014)
Activity of enzyme
AChE	Increased	(Schulpis et al. 2006)
BuChE	Decreased	(Stefanello et al. 2003, 2005)
Na(+),K(+)-ATPase	Decreased	(Wyse et al. 2002; Machado et al. 2011; Stefanello et al. 2003; Streck et al. 2001, 2002a, 2002b)
Inflammatory profile
IL-6	Increased	(Zhang et al. 2012)
TNF-α	Increased	(Zhang et al. 2012)

Concluding Remarks

The present review provides solid data indicating that treated and especially non-treated homocystinuric patients are susceptible to oxidative stress as evidenced by altered biomarkers that reflect lipid, protein, and DNA oxidative damage in various tissues. Additional findings obtained from experimental animal models of homocystinuria reinforce the presumption that the disruption of redox homeostasis in this disorder is induced by Hcy. Thus, it is presumed that oxidative stress may be involved and represent an important pathomechanism in the pathogenesis of the cardiovascular and neurological damage characteristic of this disease (Fig. 2). It seems therefore justified to carry out clinical studies in homocystinuric patients in order to evaluate the benefits of antioxidant supplementation in association with the mainstream therapy for these patients.

Fig. 2.

Role of oxidative stress in homocystinuria. ROS reactive oxygen species, RNS reactive nitrogen species. The figure was adapted from Servier Medical Art (www.servier.com)

Abbreviations

4-HNE

4-hydroxyalkenal

AChE

Acetylcholinesterase

ALT

Alanine aminotransferase

ApoA-1

Apolipoprotein A1

AST

Aspartate aminotransferase

BuChE

Butyrylcholinesterase

CAT

Catalase

CBS

Cystathionine ß-synthase

DCF

2′,7′-dichlorofluorescein

EC-SOD

Extracellular superoxide dismutase

G6PD

Glucose 6-phosphate dehydrogenase

GPx

Glutathione peroxidase

GRed

Glutathione reductase

GSH

Glutathione

GST

Glutathione S-transferase

H₂O₂

Hydrogen peroxide

Hcy

Homocysteine

Hhcy

Hyperhomocysteinemia

IEM

Inborn errors of metabolism

IFN-γ

Interferon-γ

IL-1β

Interleukin-1β

IL-6

Interleukin-6

MDA

Malondialdehyde

Met

Methionine

MTHFR

Methylenetetrahydrofolate reductase

O₂⁻

Superoxide anion

PKA

Protein kinase A

PON1

Paraoxonase

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

SOD

Superoxide dismutase

TAR

Total antioxidant reactivity

TAS

Total antioxidant status

TBARS

Thiobarbituric acid reactive substances

TRAP

Total radical-trapping antioxidant potential

TUNEL

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling

Vitamin B6

Pyridoxine