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Journal of the American College of Nutrition, Vol. 23, No. 5, 386-390 (2004)
Published by the American College of Nutrition

Hyperhomocysteinemia, Enzyme Polymorphism and Thiobarbituric Acid Reactive System in Children with High Coronary Risk Family History

Tamas Szamosi, MD, PhD, FACN, Erzsebet Roth, MD, PhD, Tamas Szamosi, Jr, MD, Erika Tomsits, MD, PhD, Attila Tordai, MD and Terez Szabo, MD, PhD

2nd Department of Pediatrics, Medical Faculty, Semmelweis University (T.S., E.T.), Budapest, HUNGARY
Department of Research Surgery, Medical Faculty, University of Pecs (E.R.), Budapest, HUNGARY
National Health Center (T.S.Jr., A.T.), Budapest, HUNGARY
Department of Clinical Pathology, Heim Pal Children’s Hospital (T.S.), Budapest, HUNGARY

Address reprint requests to: Tamas Szamosi, M.D., Ph.D., 2nd Department of Pediatrics, Med. Fac. Semmelweis Univ., Tüzoltó u. 7, H-1094 Budapest, HUNGARY. E-mail: szatam{at}gyer2.sote.hu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
 REFERENCES
 
Objectives: To investigate both the frequency and the genetic background of hyperhomocysteinemia and the frequency of increased plasma thiobarbituric acid reactive system (TBARS) levels in children and adolescents whose parents had premature coronary heart disease (CHD).

Methods: The study was performed on children and adolescents aged 4–18 years (105 offspring of parents with CHD before age 45 and 74 referents from families without any evidence of premature atherosclerosis). Fasting serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and total triglyceride (TG) levels were measured by enzymatic methods. Low density lipoprotein cholesterol (LDL-C) level was calculated by the Friedewald formula. Plasma total homocysteine (THCy) level was measured by fluorescence polarisation immunoassay. Plasma TBARS level was determined by fluorimetric method. 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme polymorphism was analyzed by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP).

Results: Hyperhomocysteinemia was found in 32 cases and in 4 controls. Increased plasma THCy level was found in 10 children and adolescents from 12 cases homozygous for the C677T polymorphism of the MTHFR gene. No similar high frequency was observed in heterozygous subjects. Elevated fasting plasma TBARS levels were found in 38 cases and in 8 controls. The frequency differences were significant (p < 0.01). Allele frequency of the MTHFR polymorphism among cases and controls was similar. Significant correlation (r = 0.53, p < 0.02) was detected between plasma THCy and TBARS levels. One child had high serum TC level, 5 had low serum HDL-C level and all other children had normal serum TC, LDL-C, HDL-C and TG levels from children with hyperhomocysteinemia and/or high plasma TBARS levels. A significant correlation (r = 0.64, p < 0.01) was observed between plasma THCy levels of parents and children in the case group.

Conclusion: The measurement of plasma THCy and TBARS levels may contribute to the detection of the risk of children and adolescents with high CHD risk family history.

Key words: plasma total homocysteine, plasma TBARS, children, parents, CHD


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 CONCLUSION
 REFERENCES
 
Atherosclerosis begins in childhood [1]. Pathologic data have shown that the extent of atherosclerotic change in children can be correlated with the presence of the same risk factors identified in adults [2]. In controlled studies risk factors of atherosclerosis as the high serum total cholesterol (TC), and lipoprotein (a), low high density lipoprotein cholesterol (HDL-C) and hypertension were found by numerous authors in children and adolescents of parents with premature coronary heart disease (CHD) [3–5]. High plasma thiobarbituric acid reactive system (TBARS) level as a sign of the increased lipid peroxidation was observed in children of families with accumulated premature CHD (for example, father and grandfather had premature CHD) [6]. These results suggest searching for further risk factors which may promote the atherosclerotic process in children and adolescents with a high risk atherosclerotic family history.

Hyperhomocysteinemia is also suggested to be a risk factor of atherosclerosis [7–9]. Plasma total homocysteine (THCy) level is affected by genetic factors and nutrition. B6, B12 vitamine and folate deficiency can cause high plasma THCy level [10,11]. Folate and B6 vitamine therapy may diminish the plasma THCy level in hyperhomocisteinemia combined enzyme polymorphism, as well [12]. The significance of genetic factors including the widely investigated 5–10 methylenetetrahydrofolate reductase (MTHFR) enzyme C677T polymorphism has been discussed [13–15]. The common effect of hyperhomocysteinemia and the low density lipoprotein (LDL) modification by free oxygen radicals on vessel wall cells was described [16]. One of the methods for the estimation of the possible modification rate of the LDL is the measurement of plasma thiobarbituric acid reactive system (TBARS) level in plasma [17].

The diet is an important part of the preventive care of children having atherosclerotic risk factors. In order to make effective dietetic decisions the knowledge of every possible risk factor is important. Hyperhomocysteinemia and high plasma TBARS levels are suggested to be risk factors of atherosclerosis affected by a not usually low fat, low cholesterol diet. According to these results the investigation of the frequency of hyperhomocysteinemia and high plasma TBARS levels in children and adolescents whose parents had early CHD seems to be useful.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 CONCLUSION
 REFERENCES
 
Children and adolescents (58 boys, 47 girls, altogether 8 siblings, aged 4–18 years) whose parents had premature acute myocardial infarction (AMI) (proven by ECG and enzyme investigations) before age 45 were investigated together with age matched referents (36 boys, 38 girls, among them 6 siblings) who arrived at the Pediatric Department for adenoidectomy or tonsillectomy without any evidence of premature atherosclerotic disease in their families. Informed consent was obtained from all parents and schoolchildren.

Blood was collected from the cubital vein. Fasting serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), total triglyceride (TG) levels were measured by enzymatic methods using Boehringer-LaRoche kits. Serum LDL-cholesterol (LDL-C) was calculated by Friedewald formula [18], because no serum TG level above 400 mg/dL was detected in any cases, or controls. Plasma THCy level was measured by the IMX (Abbott) fully automatic method with a reduction step to make the plasma THCy free after which it was converted to S-adenosyl-homocysteine (SAH) followed by a competitive immunoassay using anti-SAH antibody and a fluorescent tracer analog. [19]. Hyperhomocysteinemia was defined above the pediatric reference ranges described by Vilaseca et al. [20]. Deoxyribonucleic acid (DNA) polymorphism (C677T) of the MTHFR enzyme was examined by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) [21] using genomic DNA extracted from peripheral blood samples by the salting-out technique. Plasma TBARS was determined by fluorimetric method of Yagi [17]. Plasma for TBARS and THCy determinations was stored in different tubes.

Statistical analysis was made by {chi}2 probe for the frequency analysis and by a statistical software (ANOVA, Fisher-Exact test). A 0.05 value of the p was defined as significance limit.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
CONCLUSION
 REFERENCES
 
Increased plasma THCy levels (11.3–33.5 µmol/L) were observed in 32 high risk and in 4 referent children or adolescents. Increased plasma TBARS levels (2.5–6.9 nmol/mL) were found in 38 high risk and in 8 referent children. These differences were statistically significant (Table 1). Significant correlation was found between plasma THCy and TBARS levels (Fig. 1).


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  		Table 1. The Frequency of Increased and Normal Plasma THCy and TBARS Levels in the Different Groups

 


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  		Fig. 1. Correlation between plasma THCy and TBARS levels of children and adolescents (r = 0.53, p < 0.02).

 
According to the frequency analysis of the C677T mutation of the MTHFR gene in 36 children with hyperhomocysteinemia 12 (31%) wild type, 14 (40%) heterozygous and 10 (29%) homozygous genotypes for this polymorphism were found. The allele frequency was 48% which did not differ significantly from the frequency (32%) found in 73 offspring and 70 referents without hyperhomocysteinemia. The frequency analysis of the C677T mutation of the MTHFR gene in these children and adolescents with normal plasma THCy levels showed 54 (37.8%) wild type, 87 (60.8%) heterozygous and 2 (1.4%) homozygous genotypes (Table 2). Genotype frequencies were in agreement with the Hardy-Weinberg expectations in all populations.


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  		Table 2. C677T Mutation of the MTHFR Gene in Subjects with Different THCy Levels

 
The mean THCy level of the 12 homozygous children and adolescents was 19.8 µmol/L, significantly higher than that of subjects (7.5 µmol/L) belonging to the wild type group. No similar significant difference was observed between children and adolescents belonging to the heterozygous (9.7 µmol/L) or the wild type group (Table 3).


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  		Table 3. Mean Plasma THCy Levels of Subjects Belonging to Different Genotypes for the C677T Mutation of the MTHFR Gene

 
Increased serum TC levels (5.2–7.4 mmol/L) were observed in 38 high risk and in 3 referent children or adolescents. Increased serum LDL-C levels (3.5–6.3 mmol/L) were found in 37 high risk and 4 referent children. Decreased serum HDL-C levels (0.6–0.9 mmol/L) were observed in 38 high risk and in 6 referent children. These frequency differences were statistically significant. No significant differences were calculated in serum TG levels (Table 4).


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  		Table 4. The Frequency of Abnormal and Normal Serum Lipid Levels in the Different Groups

 
Increased serum TC and LDL-C level together with increased plasma THCy level was found in one high risk case. Decreased serum HDL-C level together with elevated plasma THCy and TBARS levels were observed in 4 high risk cases and in one control.

Significant correlation was found between the plasma THCy levels of parents with early CHD and that of their children (Fig. 2).



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  		Fig. 2. Plasma THCy levels (µmol/L) in high risk families (r = 0.64, p < 0.01).

 

   DISCUSSION
 
Frequent occurrence of increased plasma THCy and/or TBARS levels was found in children and adolescents with high coronary risk family history. According to this result preventive care should be emphasized by modifying and controlling factors influencing plasma THCy and TBARS level included B6 vitamine, folate [11] and/or antioxidant trace element rich diet.

MTHFR gene polymorphism accounts for the risk of a family history for AMI according to Margaglione et al. [15]. Hyperhomocysteinemia found by us in our cases (children and adolescents of parents with early AMI) homozygous for the C677T polymorphism of MTHFR gene may support their opinion, because the high plasma THCy level is suggested as a risk factor of the atherosclerotic vascular disease [8].

A younger age (45 years instead of 55 years) was defined as a limit of the premature or early AMI in our study, younger than usual for practical reasons (we had the opportunity to investigate only 4–18 year old children and adolescents whose parents were younger than 45) and an easier investigation of the genetic effects of CHD described by Berg [22].

Oxidative stress is present in CHD, and hyperhomocysteinemia, an independent risk factor for this disease, may play a role by inducing production of oxygen free radicals [23]. Among the compounds with terminal carbonyl groups that result from lipid peroxidation, malondialdehyde (MDA), which is widely used as an index of oxidative damage [17,24], can interact with lipoproteins. In plasma, MDA exists as both the free form and bound to enzymes, amino acids, nucleic acids and proteins. Total MDA (free and bound) is usually determined from its reaction with thiobarbituric acid [25]. The observed correlation between plasma THCy and TBARS levels in high risk children may reinforce the role of hyperhomocysteinemia in the production of oxygen free radicals [23].

Frequent occurrence of increased serum TC and LDL-C and decreased serum HDL-C levels in the high risk children was found similarly described by others [26], but only one case had increased TC level and 4 cases (and one control) had decreased serum HDL-C level together with hyperhomocysteinemia and/or increased plasma TBARS level. This result may reinforce the independent atherosclerosis risk factor role of increased plasma THCy and TBARS levels suggested by others [27].


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
REFERENCES
 
The determination of the plasma THCy and TBARS levels seems to be an important part of the investigation of children and adolescents with high cardiovascular risk family history in order to make correct decisions about their care.

Received January 14, 2003. Revised November 11, 2003.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
 REFERENCES
 

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This Article
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