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. 2015 Mar 25;35(6):899–911. doi: 10.1007/s10571-015-0185-7

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

Camila Simioni Vanzin 1,2,✉, Caroline Paula Mescka 1,2, Bruna Donida 2,5, Tatiane Grazieli Hammerschimidt 2, Graziela S Ribas 2, Janaína Kolling 1, Emilene B Scherer 1, Laura Vilarinho 3, Célia Nogueira 3, Adriana Simon Coitinho 4, Moacir Wajner 1,2, Angela T S Wyse 1, Carmen Regla Vargas 1,2,5,✉
PMCID: PMC11486249  PMID: 25805165

Abstract

Cystathionine-β-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.

Keywords: Homocysteine, Lipid profile, Inflammation, Paraoxonase, Butyrylcholinesterase, Vitamin B12, Folic acid

Introduction

Homocystinuria is a metabolic disorder characterized by the accumulation of the amino acid homocysteine (Hcy) in biological fluids of affected patients. Cystathionine β-synthase (CBS) deficiency is the most frequently encountered cause of homocystinuria. In addition to Hcy, methionine (Met) and a variety of other metabolites of Hcy accumulate in the body or are excreted in the urine of such patients. Dislocation of the optic lens, osteoporosis, thinning and lengthening of the long bones, mental retardation, and thromboembolism affecting large and small arteries and veins are the most common clinical features. However, thromboembolism represents the major cause of morbidity and death in CBS-deficient patients. It is unclear the cause of the cardiovascular changes found in patients with homocystinuria due to CBS deficiency, but evidence suggests that blood coagulation disorders, damage to lipoproteins, or effects on the platelets, on the endothelium and the non-vascular endothelial cells may be involved (Mudd et al. 2001).

Management of CBS-deficient patients leads to amelioration of the characteristic biochemical abnormalities (Mudd et al. 2001). Recognized modalities of treatment include pyridoxine (vitamin B6) in combination with folic acid and vitamin B12, methionine-restricted diet, cystine-supplemented diet, and betaine (Yap 2003). Treatment of patients with vitamin B6 in combination with folate or betaine lowers plasma total homocysteine (tHcy) and improves vascular outcomes, suggesting that Hcy or its metabolites play a causal role in aterothrombosis (Yap et al. 2001). Hcy itself, S-adenosyl-Hcy, Hcy-thiolactone, and Hcy-thiolactone-modified protein (N-Hcy-protein) are potential candidates, and possible mechanisms have been assigned to each (Yap et al. 2001; Jakubowski 2001, 2007; Perla-Kaján et al. 2007). Hcy-thiolactone is a reactive metabolite that causes protein N-homocysteinylation what impairs or alters the protein’s function (Jakubowski 2008). It was observed that Hcy-thiolactone and N-Hcy-protein are elevated in patients with CBS deficiency (Jakubowski et al. 2008). Paraoxonase (PON1), a calcium-dependent enzyme carried on HDL in the blood, hydrolyzes Hcy-thiolactone and protects against the accumulation of N-Hcy-protein in vivo and in vitro (Jakubowski et al. 2000; 2001; Perla-Kaján and Jakubowski 2010).

Butyrylcholinesterase (BuChE) is an α-glycoprotein synthesized in the liver. Its enzymatic activity is positively associated with cardiovascular risk factors. Several investigators have found significant relationships between cholinesterase activity and triacylglycerols, HDL cholesterol, and LDL cholesterol (Santarpia et al. 2013). Additionally, the knowledge that chronic inflammation is implicated in the pathogenesis of atherosclerosis established the use of inflammatory markers for assessing coronary risk. Among these biomarkers are pro-inflammatory cytokines such as IL-1, IL-6, and INF-γ (Pearson et al. 2003).

We recently demonstrated that lipid and protein oxidative damage is increased and that antioxidant defenses are reduced in plasma of CBS-deficient patients, probably due to the increase in reactive species generation induced by Hcy (Vanzin et al. 2011). Furthermore, it is known that the enzymes PON1 and BuChE contain SH-groups which are important for their activities (Nishio and Watanabe 1997; Aviram et al. 1998) and that Hcy is able to induce alterations in BuChE activity in vivo and in vitro, as demonstrated by Stefanello et al. (2003, 2005). In this context, the aim of this study was to evaluate lipid (total cholesterol, HDL cholesterol, LDL cholesterol, oxidized LDL cholesterol, apolipoprotein A-1) and inflammatory profile (IL-1β, IL-6, and INF-γ), as well as protein oxidative damage and the activities of the enzymes PON1 and BuChE in plasma of patients with homocystinuria due to CBS deficiency, analyzing the effect of the treatment, especially of folic acid and vitamin B12, on these parameters. Additionally, we correlated all parameters with the total Hcy, folic acid, and vitamin B12 levels.

Materials and Methods

Patients and Controls

The present study was approved by the Ethics Committee of Hospital de Clínicas de Porto Alegre, RS, Brazil. Informed consent was obtained according to the guidelines of the committee, number 100290.

Patients

Subjects with homocystinuria due to CBS deficiency were recruited from the Medical Genetic Service of Hospital de Clínicas de Porto Alegre, Brazil. Sex, age, and metabolic features of patients are described in Table 1. Plasma samples were obtained from 10 patients at the moment of diagnosis (median age 10 years; range 4–27 years) (Group A) and 10 patients under treatment (median age 19 years; range 12–32 years) (Group B). All patients were diagnosed after the neonatal period by identification of abnormal elevated concentrations of tHcy and Met in plasma. The major clinical manifestations were ectopia lentis, seizures, developmental delay, thinning and lengthening of the long bones (marfanoid appearance). The prescribed treatment consisted of a protein-restricted diet supplemented by pyridoxine (median dose: 500 mg/day; range: 100–750 mg/day), folic acid (median dose: 5 mg/day; range 2–5 mg/day), betaine (median dose: 6 g/day; range 2–6 g/day), and vitamin B12 (median dose: 1 mg IM/month). The average duration of treatment was 10 years (range 5–20 years).

Table 1.

Clinical and metabolic features of CBS-deficient patients

Patient Sex Age at diagnosis (years) tHcy (µmol/L) Met (µmol/L)
1* M 10 238.6 108.3
2* M 10 189.08 551.1
3* M 4 195.38 650.4
4* F 9 354.5 277.0
5* F 10 318.42 581.3
6* F 10 341.81 377.5
7* M 5 316.72 929.5
8* F 27 245.7 230.5
9* F 8 197.9 95.3
10* M 14 393.7 599.3
11 M 12 135.82 60.4
12 M 14 179.6 735.7
13 M 15 29.3 42.0
14 M 19 164.4 822.6
15 F 23 264.8 75.8
16 F 19 48.4 539.1
17 F 28 240.61 66.0
18 M 32 280.96 141.0
19 M 20 165.25 64.2
20 M 18 24.01 21.2
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* Patients with homocystinuria at diagnosis (untreated)

Controls

Healthy individuals with comparable age and sex of the patients were recruited from the Laboratório de Análises Clínicas da Universidade Federal do Rio Grande do Sul. Plasma samples were obtained from 13 individuals (median age: 25 years; range 4–34 years).

Plasma Preparation

Plasma was separated from whole blood samples obtained from controls and CBS-deficient patients by venous puncture with heparinized vials. Whole blood was centrifuged at 3000×g for 10 min at 4 °C; plasma was removed by aspiration and frozen at −80 °C until analysis.

Lipid Profile

Total Cholesterol Levels

Total cholesterol was measured by a commercial kit (Labtest Diagnóstica, Minas Gerais, Brazil). The results were expressed as mg/dL.

HDL Cholesterol Levels

HDL cholesterol levels were measured by a commercial kit (Labtest Diagnóstica, Minas Gerais, Brazil). The system uses two reagents which enable selective dosage of cholesterol bound to HDL. The results were expressed as mg/dL.

Triglycerides Levels

Triglycerides levels were evaluated by a commercial kit (Labtest Diagnóstica, Minas Gerais, Brazil). The results were expressed as mg/dL.

LDL Cholesterol Levels

The LDL levels were calculated through the Friedewald formula:

LDL cholesterol=Total cholesterol-HDL cholesterol-Triglycerides5.

The results were expressed as mg/dL.

Oxidized LDL Cholesterol Levels

Oxidized LDL cholesterol levels were measured by a commercial kit (Mercodia, Sweden). Sandwich ELISA based on the mouse monoclonal antibody 4E6, which is specific for a conformational epitope in oxidized ApoB-100, was utilized. The results were expressed as U/L.

Apolipoprotein A1 (ApoA-1)

ApoA-1 levels were evaluated by a commercial kit (BioTécnica, Brazil). The determination is based on an immunoturbidimetric method in which antibodies specific to apolipoprotein A1 form an insoluble complex, and the turbidity produced is proportional to the amount of ApoA1 in sample. The results were expressed as mg/dL.

Inflammatory Profile

Plasma IL-1β, IL-6, and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA) kits (Mabtech AB, Sweden). The assay utilizes ELISA strip plates pre-coated with a capture monoclonal antibody (mAb), to which samples are added. Captured cytokine is detected by adding a biotinylated mAb followed by streptavidin-horseradish peroxidase. Addition of the enzyme substrate TMB results in a colored product. Intensity of the color is directly proportional to the concentration of cytokine in the sample, which is determined by comparison with a serial dilution of recombinant cytokine standard analyzed in parallel. The results were expressed as pg/mL.

Paraoxonase Activity (PON1)

PON1 enzyme activity was assessed according to Eckerson et al. (1983). Initially, it was prepared a solution containing 4.8 mL buffer glycine/NaOH pH 10.6, 0.9 mM CaCl2, and 1.06 mL of 1.0 mM paraoxon. For each sample, it was utilized 780 µL of this solution and 20 µL of plasma. The absorbance was measured in a spectrophotometer at 412 nm in three times with intervals of 1 min, thus obtaining the release of para-nitrophenol per minute. The blank was assayed the same way, but without the sample, and was subtracted from the absorbances obtained. The results were expressed as U/mL (1 U of enzyme hydrolyzes 1 µmol of paraoxon per minute).

Butyrylcholinesterase Activity (BuChE)

BuChE activity was determined by the method of Ellman et al. (1961) with some modifications. Hydrolysis rate was measured at acetylthiocholine concentration of 0.8 mM in 1 mL assay solutions with 100 mM potassium phosphate buffer pH 7.5 and 1.0 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB). Fifty microliters of diluted plasma was added to the reaction mixture and preincubated for 3 min. The hydrolysis was monitored by formation of the thiolate dianion of DTNB at 412 nm for 2 min (intervals of 30 s) at 25 °C. All samples were run in duplicate. Specific enzyme activity was expressed as mmol acetylthiocholine hydrolyzed per hour per milligram of protein.

Total Homocysteine (tHcy) Measurement

The total homocysteine levels in plasma were measured by liquid chromatography electrospray tandem mass spectrometry (LC–MS/MS), as described by Magera et al. (1999). This method is based on the analysis of 100 µL of plasma with 20 µL of homocysteine-d8 (2 nmol) added as internal standard. After the step of reduction with 20 µL of 500 mM dithiothreitol followed by deproteinization, the analysis was performed in the multiple reaction monitoring mode in which total Hcy and Hcy-d4 were detected through the transition from the precursor ion (m/z 136–m/z 90 and m/z 140–m/z 94, respectively). The retention times of total Hcy and Hcy-d4 were 1.5 and 2.5 min, respectively. The calibration was performed by a curve with 5 concentrations of Hcy. The results were expressed as μmol/L. In plasma, tHcy is the sum of free and protein-bound homocysteine, homocystine, and several other mixed disulfides.

Folic Acid Measurement

Folic acid levels were measured in plasma by electrochemiluminescence using the analyser Elecsys 2010 (Roche Diagnostics GmbH, Mannheim, Germany). The results were expressed as ng/mL.

Vitamin B12 Measurement

Vitamin B12 levels were measured in plasma by electrochemiluminescence using the analyser Elecsys 2010 (Roche Diagnostics GmbH, Mannheim, Germany). The results were expressed as pg/mL.

Sulphydryl Groups Content

This assay is based on the reduction of 5.5′-dithiobis (2-nitrobenzoic acid) (DTNB) by thiols, generating a yellow derivative whose absorption is measured spectrophotometrically at 412 nm (Aksenov and Markesbery 2001). Results were reported as nmol TNB.

Carbonyl Content

Carbonyl content was measured according to the method described by Levine et al. (1990). Briefly, duplicate aliquots of plasma (100 µL) were treated with 100µL of trichloroacetic acid 28 %. The tubes were centrifuged at 8000×g for 10 min to obtain the protein pellet. One milliliter of 2.4-dinitrophenylhydrazine (DNPH) 10 mM prepared in 2 M HCl or 1.0 mL of 2 M HCl (blank) were added to the precipitates and incubated at 37 °C for 90 min. After, the samples were centrifuged and the DNPH excess was removed with ethanol–ethyl acetate 1:1 (v/v). The final protein pellet was dissolved in 200µL of 6 M guanidine hydrochloride. Quantification was performed using a spectrophotometer at 370 nm. The carbonyl content was calculated using a millimolar absorption coefficient of the hydrazone (21.000 M−1 cm−1). Values of carbonyl content were expressed in nmol carbonyl/mg protein.

Protein Determination

Protein was measured by the method of Bradford (1976), using serum bovine albumin as standard.

Statistical Analysis

Data were expressed as mean ± standard deviation. Comparisons between means were analyzed by one-way ANOVA followed by the Duncan multiple range test when the F value was significant. Correlations between variables were calculated using the Pearson correlation coefficient. A p value lower than 0.05 was considered significant. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software in a PC-compatible computer.

Results

In this study, we evaluated the lipid and inflammatory profiles, protein oxidation, and the activities of enzymes PON1 and BuChE in plasma of CBS-deficient patients at diagnosis (group A) and under treatment (group B). These parameters were compared to those of controls with similar ages. Furthermore, we correlated all parameters measured with the tHcy, folic acid, and vitamin B12 concentrations.

With regard to lipid profile (Table 2), we found a significant decrease in HDL [F(2.26) = 9.60, p < 0.001] and apolipoprotein A1 [F(2.29) = 13.00, p < 0.001] levels in both groups of CBS-deficient patients (at diagnosis and under treatment) when compared to controls. LDL and oxidized LDL levels were statistically similar in all groups. Total cholesterol levels were significantly reduced in both groups of CBS-deficient patients [F(2.25) = 6.88, p < 0.01], probably due to the decrease in HDL levels. Total Hcy levels were significantly increased in patients at diagnosis and during treatment, showing that the therapy approach was not able to adequately control the tHcy levels. Sulphydryl groups content was significantly decreased in both treated or not treated patients [F(2.23) = 4.94, p < 0.05].

Table 2.

Lipid profile, total Hcy levels, and sulphydryl content in CBS-deficient patients at diagnosis (Group A), CBS-deficient patients under treatment (Group B) and controls

Controls Group A Group B
Total cholesterol (mg/dl) 185.62 ± 56.53 118.75 ± 38.48* 125.78 ± 28.36*
HDL cholesterol (mg/dl) 59.35 ± 19.43 38.98 ± 9.62** 33.27 ± 9.99**
LDL cholesterol (mg/dl) 106.65 ± 53.95 71.14 ± 30.45 78.76 ± 29.21
Oxidized LDL cholesterol (U/L) 19.15 ± 7.44 14.77 ± 4.76 13.12 ± 5.75
Apolipoprotein A (mg/dL) 200.98 ± 29.57 156.32 ± 33.81** 141.34 ± 20.79**
tHcy (µmol/L) 5.84 ± 2.52 266.5 ± 66.71*** 137.8 ± 104.3*
Sulphydryl content (nmol TNB) 384.91 ± 82.78 247.99 ± 92.52# 282.66 ± 112.11#
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Data represent mean ± standard deviation

# p < 0.05 Statistically different from controls (ANOVA, followed by the Duncan multiple range test)

* p < 0.01 Statistically different from controls (ANOVA, followed by the Duncan multiple range test)

** p < 0.001 Statistically different from controls (ANOVA, followed by the Duncan multiple range test)

*** p < 0.0001 Statistically different from controls (ANOVA, followed by the Duncan multiple range test)

Additionally, the activities of enzymes PON1 and BuChE were evaluated. PON1 activity was decreased in both groups A and B of CBS-deficient patients when compared to controls [F(2.27) = 11.73, p < 0.001] (Fig. 1). Otherwise, BuChE activity was increased only in the group of untreated patients when compared to controls and to the treated CBS-deficient patients [F(2.25) = 7.72, p < 0.01] (Fig. 2). We verified significant positive correlations between PON1 activity and sulphydryl groups content (r = 0.596, p < 0.05) (Fig. 3) and, as expected, between HDL and apolipoprotein A-1 levels (r = 0.589, p < 0.05) (Fig. 4).

Fig. 1.

Fig. 1

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PON1 activity in CBS-deficient patients at diagnosis (Group A, n = 9), CBS-deficient patients under treatment (Group B, n = 9) and controls (n = 12). Data represent mean ± standard deviation, ** p < 0.001 statistically different from controls (ANOVA, followed by the Duncan multiple range test)

Fig. 2.

Fig. 2

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BuChE activity in CBS-deficient patients at diagnosis (Group A, n = 8), CBS-deficient patients under treatment (Group B, n = 8) and controls (n = 11). Data represent mean ± standard deviation, *p < 0.01 statistically different from controls and from group B (ANOVA, followed by the Duncan multiple range test)

Fig. 3.

Fig. 3

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Correlation between PON1 activity and sulphydryl groups content (r = 0.596, p < 0.05) in plasma from CBS-deficient patients at diagnosis and under treatment

Fig. 4.

Fig. 4

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Correlation between HDL levels and apolipoprotein A-1 levels (r = 0.589, p < 0.05) in plasma from CBS-deficient patients at diagnosis and under treatment

Next, we evaluated the inflammatory profile in both groups of CBS-deficient patients (Table 3). We demonstrated that IL-6 was significantly higher in group A when compared to controls [F(2.26) = 3.897, p < 0.05]. It was verified a tendency to reduction (27.68 %) in the IL-6 levels in group B when compared to group A. Similar results were observed in carbonyl groups formation, which were significantly higher in group A when compared to controls [F(2.21) = 3.565, p < 0.05], presenting a tendency to reduction (15,2 %) in group B when compared to group A. We found a significant positive correlation between IL-6 levels and carbonyl groups content (r = 0.551, p < 0.05) (Fig. 5), indicating a possible association between inflammation and oxidative protein damage in plasma of CBS-deficient patients. Additionally, we also evaluated the IL-1β and INF-γ levels which were statistically similar in all groups (patients and controls).

Table 3.

Inflammatory profile and carbonyl content in CBS-deficient patients at diagnosis (Group A), CBS-deficient patients under treatment (Group B) and controls

Controls Group A Group B
Interleukin-6 (pg/mL) 11.74 ± 3.54 22.87 ± 9.81* 16.54 ± 9.16
Interleukin-1β (pg/mL) 19.47 ± 12.49 17.23 ± 12.41 15.99 ± 11.63
Interferon-γ (pg/mL) 50.22 ± 12.91 51.40 ± 16.15 59.46 ± 25.16
Carbonyl content (nmol/mg protein) 0.278 ± 0.038 0.355 ± 0.091* 0.303 ± 0.366
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Data represent mean ± standard deviation

* p < 0.05 statistically different from controls (ANOVA, followed by the Duncan multiple range test)

Fig. 5.

Fig. 5

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Correlation between IL-6 levels and carbonyl groups content (r = 0.551, p < 0.05) in plasma from CBS-deficient patients at diagnosis and under treatment

Finally, we correlated all parameters investigated with the folic acid and vitamin B12 concentrations. We found a significant positive correlation between vitamin B12 and apolipoprotein A-1 levels (r = 0.535, p < 0.05) (Fig. 6), as well as a significant positive correlation between vitamin B12 levels and PON1 (r = 0.631, p < 0.05) (Fig. 7). Additionally, we demonstrated a significant negative correlation between folic acid and total Hcy concentrations (r = −0.633, p < 0.05) (Fig. 8). Folic acid was increased in treated patients (16.59 ± 5.03 ng/mL; mean ± standard deviation) when compared with not treated patients (7.85 ± 5.11 ng/mL; mean ± standard deviation) [t(18) = 3.541, p < 0.01], as expected. On the other hand, vitamin B12 levels were similar in both groups of patients (299,25 ± 150,88 pg/mL before treatment versus 330,32 ± 185,68 pg/mL after treatment; mean ± standard deviation) [t(21) = 0.964, p > 0.05], indicating that these patients presented a poor adherence to the treatment.

Fig. 6.

Fig. 6

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Correlation between vitamin B12 levels and apolipoprotein A-1 levels (r = 0.535, p < 0.05) in plasma from CBS-deficient patients at diagnosis and under treatment

Fig. 7.

Fig. 7

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Correlation between vitamin B12 levels and PON1 (r = 0.631, p < 0.05) in plasma from CBS-deficient patients at diagnosis and under treatment

Fig. 8.

Fig. 8

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Correlation between folic acid and total Hcy levels (r = −0.633, p < 0.05) in plasma from CBS-deficient patients at diagnosis and under treatment

Discussion

A link between Hcy and atherothrombotic vascular disease was first suggested by McCully (1969) more than 40 years ago. Since that time, many investigations have been conducted to define the cause(s) of atherosclerosis in CBS-deficient patients. Findings suggest that Hcy or its derivatives other than Met (for example homocysteine-thiolactone) may be the major contributor(s) to the vascular damage of CBS deficiency (Mudd et al. 2001). In this sense, we evaluated in this work the lipid (total cholesterol, HDL cholesterol, LDL cholesterol, oxidized LDL cholesterol, apolipoprotein A1) and the inflammatory (IL-6, IL-1β, IFN-γ) profiles, protein oxidative damage (sulphydryl and carbonyl groups content), and the activities of enzymes PON1 and BuChE in plasma of CBS-deficient patients, treated and not treated (at diagnosis). We also correlated these measurements with tHcy, folic acid, and vitamin B12 concentrations.

The atherogenicity of Hcy may involve several mechanisms including LDL cholesterol oxidative modification and HDL cholesterol decrease (Xiao et al. 2011). Low HDL cholesterol is a strong independent predictor of coronary artery disease (CAD) when its level are <40 mg/dL (JAMA 2001). Studies reported that Hcy is able to inhibit ApoA-1 expression and decrease HDL cholesterol levels in vitro and in vivo (animal model) (Liao et al. 2006; Mikael et al. 2006). ApoA-1 is the major protein component of HDL cholesterol and it is known that the protective effect of ApoA-1 may be related to its association with increased HDL production. Case–control studies have provided supporting data showing that plasma total Hcy was negatively correlated with HDL cholesterol levels in patients with myocardial infarction (Qujeq et al. 2001). An effect of Hcy on HDL metabolism could be clinically important, because HDL protects against vascular disease not only by facilitating reverse cholesterol transport but also through its direct anti-inflammatory properties (Devlin and Lentz 2006). In our work, we found a reduction of HDL and ApoA-1 plasma levels in CBS-deficient patients when compared to controls. An interesting fact is the failure of the treatment to prevent or reverse these reductions, since both groups of patients, at diagnosis and during treatment, presented a decrease of HDL and ApoA-1 plasma levels. It was observed in this study a significant correlation between vitamin B12 and Apo A-1 levels, as well as between vitamin B12 and PON1. These findings suggest that vitamin B12 could be essential to increase the Apo A-1 levels and PON1 activity in CBS-deficient patients, which is interesting since these components demonstrate important atheroprotective effects (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 not treated CBS-deficient patients. It is important to emphasize that probably the treated patients involved in this study have a poor adherence to the treatment with vitamin B12, which was prescribed to patients in a dose of 1 mg/month intramuscularly. More studies evaluating the effect of vitamin B12 in patients with good adherence to therapy are necessary.

Our results are different from those found by Jiang et al. (2012) who demonstrated that ApoA-1 was decreased only in not treated CBS-deficient patients. The treatment, in the patients studied by Jiang et al. was based in protein restriction in addiction to cysteine and betaine supplementation. In the present work, the patients were treated by a protein-restricted diet supplemented by pyridoxine, folic acid, betaine, and vitamin B12, without cysteine supplementation. This can partially explain the differences found in the two studies, since a previous study demonstrated that plasma levels of ApoA-1 were positively associated with plasma cysteine levels (Nuño-Ayala et al. 2010). Besides, in the work of Jiang et al. (2012), treated patients presented total Hcy concentrations between 24.5 and 86.1 µM, while the patients of our study had much higher levels of this metabolite.

It is possible that the effects observed on ApoA-1 and HDL cholesterol could be attributed to any metabolite of Hcy, such as Hcy-thiolactone. Hcy-thiolactone is a product of an error-editing reaction in protein biosynthesis which is formed when Hcy is mistakenly selected by methionyl-tRNA synthetase. Pathophysiological consequences of protein N-homocysteinylation include, among other things, protein and cell damage (Jakubowski 2008). A recent study demonstrated that N-Hcy-ApoA1 is present in humans (Ishimine et al. 2010). In this sense, it is important to emphasize that the adherence to the treatment is essential to reduce the tHcy levels and, consequently, to reduce the Hcy-thiolactone production, as well as their harmful effects. It is important to emphasize that folic acid supplementation in the patients of our study induced a decrease in tHcy levels, since a negative correlation was observed between this compound and Hcy, although this amino acid concentration remained higher than those expected to an adequate treatment.

In a recent work, we demonstrated that oxidative protein damage occurs in plasma of CBS-deficient patients at diagnosis and during the treatment probably due to the high tHcy levels (Vanzin et al. 2011). In this work, we found a significant positive correlation between sulphydryl groups content, a biomarker inversely proportional to protein oxidative damage, and PON1 activity. Moreover, we verified a reduction in PON1 activity in the both groups of CBS-deficient patients (at diagnosis and under treatment) when compared to controls. PON1, a component of HDL cholesterol, is a calcium-dependent multifunctional enzyme that connects the metabolism of lipoproteins and Hcy. Due to its ability to reduce oxidative stress, PON1 contributes to atheroprotective functions of HDL in mice and humans. Furthermore, PON1 has the ability to hydrolyze a variety of substrates, including Hcy-thiolactone (Perla-Kaján and Jakubowski 2012). In humans, Hcy-thiolactonase activity of PON1 protects against N-homocysteinylation in vivo and in vitro (Jakubowski et al. 2000; Perla-Kaján and Jakubowski 2010). In this context, we can hypothesize that the decrease in PON1 activity found in the CBS-deficient patients included in our study could be caused by Hcy-dependent oxidation of any sulfhydryl group important to the PON1 catalytic activity (Aviram et al. 1998). PON’s free sulfhydryl at cysteine-283 is required for its antioxidant activity. It is plausible, therefore, to hypothesize that the proatherogenic effects of Hcy may involve diminished serum PON1 activity, leading to impaired antioxidant function and decreased capacity to degrade Hcy-thiolactone. Furthermore, it was demonstrated that plasma tHcy remains high in patients during the treatment (137.8 ± 104.3 μmol/L; mean ± standard deviation). Additionally, as discussed before, we found a positive significant correlation between vitamin B12 levels and PON1 activity, suggesting that the fail of treatment in reversing the PON1 activity may be, at least in part, due to the insufficient doses of vitamin B12 used in the therapy, caused probably by a low adherence to the treatment. Recent studies demonstrating the effects of vitamin B12 supplementation on paraoxonase activity reinforce our hypothesis (Gu et al. 2007; Weijun et al. 2008; Koc et al. 2012). Weijun et al. (2008) demonstrated an increase of 17.59 % in HTase/PON activity in patients with type 2 diabetes after supplementation with folic acid (5 mg/day) plus vitamin B12 (500 μg/day, intramuscularly). These authors also found a significant inverse correlation between the changes in HTase/PON activity and Hcy levels. Additionally, Koc et al. (2012) found a decrease in PON activity in patients with vitamin B12 deficiency anemia, whereas PON activity was significantly increased after treatment with vitamin B12.

There is increasing evidence that acute coronary syndromes are related to activation of the immune-mediated inflammatory process associated with atherosclerotic plaques. Oxidized low-density lipoprotein (ox-LDL) is thought to play a key role in the genesis of the inflammatory process in atherosclerotic lesions (Ehara et al. 2001). In this sense, we evaluated the LDL and ox-LDL levels in plasma of both groups of CBS-deficient patients. We did not find significant differences in LDL and ox-LDL levels between patients and controls. The influence of Hcy on LDL cholesterol levels is unclear; it is known that Hcy-thiolactone can cause N-homocysteinylation in LDL, forming N-Hcy-LDL, which tends to form aggregates in vitro and induces cell death in human endothelial cells (Jakubowski 2008). Thereby, it is possible to suggest that Hcy can be acting through the formation of N-Hcy-LDL instead of ox-LDL in the CBS-deficient patients studied.

Considering that a pro-inflammatory state associated with hyperhomocysteinemia has been demonstrated by several authors (Gori et al. 2005; da Cunha et al. 2010; Keating et al. 2011), we evaluated in this study the IL-6, IL-1β, and INF-γ levels in both groups of CBS-deficient patients and controls. We found a significant increase in IL-6 levels in group A when compared to controls and a tendency to reduction (27.68 %) in the IL-6 levels in group B when compared to group A. Furthermore, we demonstrated a significant positive correlation between IL-6 levels and carbonyl groups content, indicating a possible association between inflammation and protein oxidative damage in plasma of CBS-deficient patients. Carbonyls groups are relatively difficult to induce compared with methionine sulphoxide and cysteinyl derivatives and might, therefore, indicate a more severe oxidative stress. Indeed, elevated levels of protein carbonyl are generally a sign not only of oxidative stress but also of disease-derived protein dysfunction (Dalle-Donne et al. 2003). High carbonyl content has also been observed in diabetes and arteriosclerosis and has been implicated in the accelerated vascular damage observed in these conditions (Singh et al. 2001). Additionally to IL-6, we also evaluated the IL-1β and INF-γ levels which were statistically similar in all groups (patients and controls). Our results are close to those found by Keating et al. (2011) who demonstrated an increase in IL-6 levels in homocystinuric patients before the treatment compared to treated homocystinuric patients, as well as no changes in IL-1β levels in both groups of patients.

Finally, we evaluated in this work the BuChE activity in CBS-deficient patients. It was verified that plasma BuChE activity was increased in the group of CBS-deficient patients at diagnosis when compared to controls and patients under treatment. BuChE is present in all tissues, including serum, vascular endothelia, and nervous system (Prody et al. 1987; Mack and Robitzki 2000). Preclinical studies showed that neonates rats subjected to Hcy (400–500 µmol/L) administration presented a decrease in serum BuChe activity in vitro and in vivo (Stefanello et al. 2003, 2005). Nevertheless, the effect of Hcy and its metabolites on human cholinesterases is not well understood. BuChE seems to be involved in the pathophysiology of the metabolic syndrome. Its enzymatic activity is positively associated with cardiovascular risk factors and several investigators have found significant relationships between cholinesterase activity and triacylglycerols, HDL cholesterol and LDL cholesterol (Santarpia et al. 2013). Interestingly, Darvesh et al. (2007) demonstrated that the incubation of Hcy-thiolactone with BuChE produced an immediate stimulation of the activity of this enzyme. The authors suggested that a constant stimulation of BuChE by Hcy-thiolactone could be expected to decrease acetylcholine levels. Low acetylcholine levels are known to be responsible for symptoms in Alzheimer and vascular diseases. In this work, only CBS-deficient patients at diagnosis presented increased BuChE activity, while treated CBS-deficient patients presented BuChE activity similar to controls. It is important to emphasize that the treatment based on protein-restricted diet supplemented by pyridoxine, folic acid, betaine, and vitamin B12 was able to decrease the BuChE activity, demonstrating positive results. The data of this work showing alterations in BuChE activity in plasma of CBS-deficient patients are pioneers in the literature.

In conclusion, analyzing all the aspects above discussed, we can suggest that Hcy and/or its metabolites can cause important alterations in the metabolism of CBS-deficient patients that include a decrease in PON1 activity, in HDL, and in ApoA-1 levels, as well as alterations in BuChE activity, in IL-6 levels, and induction of protein oxidative damage. These findings appear to be interconnected and correlated with protein oxidative damage. Furthermore, vitamin B12 supplementation seems to be important to improve PON1 activity and ApoA-1 levels, probably contributing to the atheroprotective effects of these components, as well as folic acid demonstrated to be essential to decrease the total Hcy levels. This work contributes to the understanding of the responsible mechanisms of vascular lesions in CBS-deficient patients and creates perspectives to future works.

Acknowledgments

This work was supported in part by grants from CAPES, CNPq, and FIPE/HCPA-Brazil. We thank immensely to the patients included in this study and to the physicians from Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre.

Conflict of interest

The authors declare that there is no conflict of interest disclosure associated with this manuscript.

Contributor Information

Camila Simioni Vanzin, Phone: (55-51) 3359 8309, Email: cami_vanzin@hotmail.com.

Carmen Regla Vargas, Phone: (55-51) 3359 8309, Email: crvargas@hcpa.ufrgs.br.

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