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Homocysteine (Hcy) is a sulfur amino acid which is an important metabolic byproduct in the methionine cycle as well as an independent risk factor in the pathogenesis of atherosclerosis,1 acute myocardial infarction,2 cerebral apoplexy,3 coronary arterial disease,4 and peripheral vascular disease.5 There is an association between the elevation of plasma Hcy level and the pathogenesis of hypertension.6 However, the pathogenesis of hypertension is not fully explained by the presence of Hcy.
Hydrogen sulfide (H2S) is a recently discovered new gasotransmitter in the modulation of cardiovascular function.7,8 H2S can be synthesized through the in vivo methionine metabolic pathway, with the catalysis by cystathionine-β-synthetase (CBS) and cystathionine-γ- lyase (CSE) of Hcy. The modulating functions of H2S on vasodilation and smooth muscle cell proliferation, resembles to a great extent, although not identical to the essential biological characteristics of nitric oxide (NO) and carbon monoxide (CO).7
In recent years, researches9,10 revealed that the reduction of the intrinsic H2S level in rats was an important factor leading to spontaneous hypertension. Since Hcy and H2S coexist in the same in vivo metabolic pathway with CSE, what would be the alterative connection between H2S and Hcy in the pathogenesis of primary hypertension? This study was aimed to investigate the significance of their metabolic imbalance in the pathogenesis of hypertension through detecting the alterations of their corresponding plasma concentration in children with primary hypertension.
METHODS
Subjects
A total of 55 children were enrolled in the study. Of them,25 children (11 boys, 14 girls) with an average age of (10.48±3.23) years who were diagnosed with primary hypertension in the Peking University First Hospital were assigned to the hypertensive group. In the hypertensive group, 20 cases were obese children, and 7 cases had a familial hypertensive history. The other 30 children (19 boys, 11 girls) with an average age of (10.57±0.73) years with normal blood pressure were assigned to the control group. Among the children in control group, 6 cases were obese children, and 2 cases had a familial hypertensive history.
The diagnostic criteria of hypertension was based on the definition which was established by the 2nd Conference of the American Children and Adolescents Blood Pressure Control Workshop in 1996. Hypertension was defined as a mean systolic blood pressure and/or mean diastolic blood pressure being equal to or over that of the 95th percentile of children and adolescents of the same age, gender, and height.11 The medical history was investigated and a physical examination and laboratory tests were performed for the two groups to exclude other diseases concerning the cardiopulmonary, urinary, and endocrine systems. An informed consent was obtained from the parents of the children and the study protocol was approved by the Ethical Committee of Peking University First Hospital, China.
General examinations
Demographic information including the patient's name, gender, age was collected and a detailed physical examination was conducted. The standard hydrargyric cuff sphygmomanometer, which met the quantitative criteria, was adopted to detect blood pressure. The environment for detection was quiet, with an optimum room temperature of 18˚C－22˚C. Any vigorous exercises, eating and drinking (except drinking water) and the intake of any medicine that might affect blood pressure were avoided at least one hour before the testing. Clothing was loose and light. The chirldren rested for 15 minutes, seated quietly.
The right brachial arterial blood pressure was detected, with the right upper limb at the same horizontal level as the heart. The width of the cuff was 2/3 the length of the upper arm, avoiding twists or folds. The lower borderline of the cuff was 2.5 cm－3 cm above the cross striation of the elbow. The bell type chest piece of the stethoscope was placed under the cuff borderline on the brachial artery at the medial side of the cubital fossa, avoiding friction against the cuff.
Before the test, the radial artery pulse was palpated. During the test, air was pumped rapidly, and the reading of the mercury column was instantly recorded when the pulse could no longer be palpated. The air was released. We waited for 1 minute and then raised the upper arm for 5－6 seconds. The air was pumped again until the mercury level reached 30 mmHg above the reading where the pulse could no longer be palpated. Air was released at a speed of 2 mmHg per second, and the blood pressure was taken down. The Korotkoff 1st, 4th, and 5th sound (muffling sound) was recorded. Three sets of consecutive testing were carried out, leaving a time interval of 1 minute after each trial and raising the upper arm for 5－6 seconds. The blood pressure records of the second and third trials were taken and their mean value was deducted. The 1st Korotkoff sound was taken as the systolic pressure while the 4th sound was the diastolic pressure.
Detection of plasma Hcy
The serum Hcy detection kit (Abbott, USA) was used along with the Abbott Irnx Automatic Immuno-analyzer, produced by the American Abbott Company. The fluorescence polarization immunoassay (FPIA) was adopted for detection. The Hcy synthesized in normal human plasma exists in the form of reductive, protein-conjugated (P-SS-Hcy) mixed disulfide compound (Cy-SS-Hcy) as well as the homocystine form (Hey-SS-Hey). Therefore, the detection method included: (1) the plasma sample being pre-treated with dithiothreitol (DDT). Hcy, the mixed disulfide form as well as the protein-conjugated form were all reduced to free Hcy (tHcy); (2) with the existence of S-adenosyl-homocysteine hydrase (SAH) and excessive adenosine, Hcy in the serum would be transformed into SAH; (3) the pre-diluted SAH mixture, anti-SAH- monoclonal antibody and the marked fluorescent S-adenosine-L-cysteine tracer were incubated simultaneously, we then employed the Abbott Irnx Automatic Immuno-analyzer to automatically detect any changes in the polarization spectrum. Finally, according to the standard curve, the quantity of Hcy in the sample could be detected.12
Detection of plasma H2S
The PXS-270 model ionic meter (Shanghai Leici, China) was employed for detection through the sensitive sulfur electrode method. H2S in the plasma usually exists in the form of gas (1/3) and NaHS (2/3). When an antioxidant is added to it, H2S and NaHS react with NaOH to give S2-. Plasma S2- was detected through the sensitive sulfur electrode in order to indirectly reflect the H2S concentration. Hence the detection procedure was as follows: (1) prepare the standard sulfur ion solution and the antioxidant solution; (2) before using the electrode, be sure it was activated for over 2 hours in de-ionized water. Switch on the ionic meter, the detection item was adjusted to “my” status and the slanting rate was adjusted to 100%; (3) both the sensitive sulfur electrode and the control electrode were immersed into the specimen, and we recorded the reading when it reached stability. The electrode should be washed with de-ionized water. After each testing, electrodes were immersed in de-ionized water to maintain its activated status. Before each testing, a standard S2- solution was used to generate the standard curve.
Statistical analysis
The SPSS 10.0 software was used for data analysis. Data were represented by mean±standard deviation (mean± SD). The t test was applied to the mean value of both groups of specimen. Pearson linear correlation analysis was applied to the plasma Hcy and H2S data as well as to the ratio of the systolic blood pressure against the plasma H2S/Hcy ratio. P<0.05 was considered statistically significant.
RESULTS
Age and blood pressure mean values of both groups
The average age of children in the hypertension group was (10.48±3.23) years old. The average systolic pressure of the group was (129.68±6.55) mmHg, and the diastolic pressure was (76.88±8.89) mmHg. The average age of children in the control group was (10.57 ±0.73) years old. Their average systolic blood pressure was (95.40±6.56) mmHg and their diastolic blood pressure was (60.60±4.64) mmHg. The average blood pressure of the two groups was significantly different (P<0.05) (Table 1).
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Table 1. Comparison of age and blood pressure between two groups (mean±SD) |
Detection of plasma Hcy in children
The average plasma Hcy in children from the hypertension group was (12.68±9.69) µmol/L, while that of the control group was (6.62±4.79) µmol/L. Plasma Hcy levels were much higher in children with hypertension than in the control group; with a t=2.996 and P<0.01 demonstrating a statistical significance between the two groups (Table 2).
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Table 2. Comparison of plasma level of Hcy, H2S and H2S/Hcy between the two groups (mean±SD) |
Detection of plasma H2S in children
Plasma H2S in children with hypertension was found to be (51.93±6.01) µmol/L, while that of the control group was (65.70±5.50) µmol/L. The plasma H2S level in children with hypertension was significantly lower than the H2S level in the control group, (t=-8.670 and P<0.01).
Ratio of plasma H2S/Hcy in both groups
The ratio of plasma H2S/Hcy in children with hypertension was found to be 5.83±2.91 while that of the control group was 11.60±3.30. The ratio of the plasma H2S/Hcy was lower in children with hypertension than in the control group, (t=-6.610 and P<0.01), and the difference is statistically significant (Table 2).
Correlation between plasma Hcy and H2S levels in both groups
Through applying the linear correlation analysis to both values, the results demonstrated a negative correlation (r=-0.379, P<0.05). That is, the higher the plasma Hcy level, the lower the plasma H2S (Fig. 1).
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Fig. 1. Correlation between plasma levels of H2S and Hcy (r=-0.379, P<0.05). The lateral axis stands for the plasma H2S level and the axis of ordinate stands for the plasma Hcy level. A correlation between plasma Hcy and H2S levels in children was created. |
Correlation between the systolic blood pressure and the value of the plasma H2S/Hcy in both groups
A linear correlation analysis was applied to compare these data. The results demonstrated an obvious negative correlation, (r=-0.687, P<0.05). That means, the higher the systolic pressure, the lower the plasma H2S/Hcy value (Fig. 2).
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Fig. 2. Correlation between systolic pressure and plasma H2S/Hcy ratio (r=-0.687, P<0.05). The lateral axis stands for the plasma H2S/Hcy level and the axis of ordinate stands for the systolic pressure. The correlation between the systolic pressure and the value of the plasma H2S/Hcy in both groups of children was created. |
DISCUSSION
Hypertension is a common disease of the cardiovascular system. Primary hypertension in children is sometimes found in adolescents. Research revealed that primary hypertension in adults may originate from childhood. A long-history of investigations of the pathogenesis of primary hypertension exists, involving vascular physiology, pathophysiology, pharmacology, cellular and molecular biology, and molecular genetics. So far, however, the pathogenesis of primary hypertension remains unclear. The elaboration of its pathogenesis is an important topic in the field which calls for urgent resolution.
Hcy is a sulfur amino acid, as well as an important metabolic by-product in the methionine metabolic pathway. Through the trans-sulfur metabolic pathway and under the action of the vitamin B6 dependent CBS, methionine condenses with serine to give cystathionine, the latter will be transformed into cysteine, α-ketosuccinic acid and NH4+ under the action of the vitamin B6 dependent CSE. Cysteine will then be further synthesized into sulfur protein, glutathione or degrade into α-ketosuccinic acid, taurine, and H2S.13 Previous studies have demonstrated that Hcy to be an independent risk factor in the pathogenesis of atherosclerosis,1 acute myocardial infarction,2 cerebral apoplexy,3 coronary arterial disease, and peripheral vascular disease.4,5
There is a correlation between an increase in plasma Hcy and the pathogenesis of hypertension.14,15 Hcy is a multi-functional injury factor.16,17 With the co-existence of transitional metal ions (Fe3+ or Cu2+), Hcy tends to undergo self-oxidation, giving rise to many strong oxidizing products (e.g. hyperoxides, hydrogen peroxides, and other peroxides) which might reduce the streaming mobility of cytoplasmic membrane and damage the intact cell, which in turn causes injury to the cellular structure and functions. The reduction in the arterial elasticity, due to alterations in arterial wall structures or functions, is an important factor in the pathogenesis of hypertension. An elevation of plasma Hcy concentration enhances the dissolution of elastic fibers and the synthesis of collagen fibers, thus altering the fixed ratio of the two fibers within the vascular wall and, in turn, altering the elasticity of the arterial wall. Hcy also enhances the proliferation of vascular smooth muscle cells, hence the middle layer smooth muscle cells of the arterial wall are increased in number and compliance decreased.
Hcy might lead to imbalance between endothelin and NO in the blood, resulting in abnormalities in both the vasoconstriction and vasodilation of the vascular endothelium. Besides, Hcy might enhance the aggregation of calcium ions inside vascular smooth muscle cells, thus promoting the elevation of vascular systolic pressure.18 The above facts indicate that homocysteinemia might play an important role in the pathogenesis of hypertension. However the pathogenic mechanism of hypertension is still not thoroughly explained.
Our research team has recently discovered that H2S, which had been regarded as a pollution gas before, turned out to be a new molecular gasotransmitter in the regulation of cardiovascular functions.8 It bears very similar though not identical biological characteristics as those of NO and CO. It might play an important role in the pathogenesis of hypertension in an animal model.14 We discovered that there is a severe inhibition of endogenous H2S synthesis in rats with spontaneous hypertension, which resulted in the thickening of blood vessels as well as the vascular structural remodeling due to proliferation of smooth muscle cells.19 After the administration of exogenous H2S, there was an obvious elevation in both the thoracic aortic H2S transformation ratio as well as plasma H2S level. This led to an obvious decrease in blood pressure, while the vascular structural remodeling was ameliorated. This indicated that a decrease of the endogenous H2S level might be an important factor of the pathogenesis of hypertension as well as aortic structural remodeling.
Through perfusion of isolated artery rings,20 we showed that exogenous H2S could help in promoting vascular dilation in hypertension. Within a certain range, it exhibited a dosage-dependent dilative reaction in the aorta of hypertensive rats while participating directly in the regulation of vasodilating functions of hypertensive rats. The above study unveils that endogenous H2S plays a very important role in maintaining the basic blood pressure as well as in the pathogenesis and development of hypertension.9 However, the significance of H2S as a new gasotransmitter in the pathogenesis of human primary hypertension is not yet fully elaborated.
The body is a very complex biological system. The maintenance of blood pressure requires preserving equilibrium among many regulatory substances, as with other in vivo homeostatic regulatory systems. In the metabolic pathway of methionine, Hcy metabolism might produce endogenous H2S under the catalysis of CBS and CSE. That means Hcy and H2S co-exist in consecutive steps adjacent to the key enzymes (CBS and CSE) of the same in vivo metabolic pathway. Hence, it could be deduced that the in vivo balance between Hcy and H2S metabolism might be a very important foundation in the homeostatic regulation of cardiovascular functions. Based on such a hypothesis, what could be the balance between these two in the pathogenesis of hypertension? Could there possibly be an imbalance of important metabolic products in the methionine metabolic pathway? Could the latter be an important mechanism of the pathogenesis of hypertension? So far, it remains unclear.
Our study discovered the mean plasma Hcy level of the hypertensive children to be (12.68±9.69) µmol/L, while that of the control group was found to be only (6.62± 4.79) µmol/L. This indicated that plasma Hcy is relatively higher in hypertensive children than in normal children (P<0.01). Moreover, the plasma H2S in children with hypertension was found to be (51.93±6.01) µmol/L, whereas that of the control group was (65.70±5.50) µmol/L, indicating that hypertensive children had lower plasma H2S level. Referring to the results of previous animal experiments, there is a decrease in the CSE expression in hypertensive rats. This indicates that in the pathogenesis of hypertension, the reduction of CSE synthesis resulted in poor metabolism of methionine and a H2S-Hcy imbalance. Since there is a decrease in plasma H2S as well as an increase in plasma Hcy in children with hypertension, it indicates that the Hcy/H2S metabolic imbalance could possibly be an important mechanism in the pathogenesis of hypertension.
Our study discovered that the Hcy/H2S imbalance in the methionine metabolism pathway plays an important role in the pathogenesis of hypertension. From this point of view, we revealed a novel idea for the pathogenesis of hypertension. However, the regulatory mechanism of the down-regulation of CSE in the vascular tissues of patients with hypertension needs further investigation.
REFERENCES
1. Kerkeni M, Addad F, Chauffert M, Chuniaud L, Miled A, Trivin F, et al. Hyperhomocysteinemia, paraoxonase activity and risk of coronary artery disease. Clin Biochem 2006; 39: 821-825. [PubMed]
2. Banu S, Mollah FH, Alam MK, Rahman MA, Hamid MA, Wahab MA, et al. Serum homocysteine concentration in patients with acute MI and chronic IHD. Mymensingh Med J 2005; 14: 54-57. [PubMed]
3. Torbus-Lisiecka B, Bukowska H, Jastrzebska M, Chelstowski K, Honczarenko K, Naruszewicz M. Lp(a), homocysteine and a family history of early ischemic cerebral stroke. Nutr Metab Cardiovasc Dis 2001; 11: 52-59. [PubMed]
4. Cattaneo M. Hyperhomocysteinaemia and atherothrombosis. Ann Med 2000; 32: 46-52. [PubMed]
5. Loncar R, Hrboka V, Tabakovic-Loncar V, Ostojic Z, Deussen A. Screening of plasma homocysteine in peripheral arterial disease. Ann Med 2001; 33: 48-54. [PubMed]
6. Yen CH, Lau YT. Vascular responses in male and female hypertensive rats with hyperhomocysteinemia. Hypertension 2002; 40: 322-328. [PubMed]
7. Tang CS, Li XH, Du JB. Hydrogen sulfide as a new endogenous gaseous transmitter in the cardiovascular system. Curr Vasc Pharmacol 2006; 4: 17-22. [PubMed]
8. Du JB, Chen XB, Geng B, Jiang HF, Tang CS. An approach on H2S as a cardiovascular molecular signal transmitter. J Peking Univ (Health Sci) (Chin) 2002; 34: 187.
9. Yan H, Du J, Tang C. The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun 2004; 313: 22-27. [PubMed]
10. Zhong G, Chen F, Cheng Y, Tang C, Du J. The role of hydrogen sulfide in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J Hypertens 2003; 21: 1879-1885. [PubMed]
11. National Heart, Lung, and Blood Institute. Update on the 1987 task force report on high blood pressure in children and adolescents: A working group report from the National High Blood Pressure Education Program. Pediatrics 1996; 98: 649-658. [PubMed]
12. Wang HL. Detection and application of homocysteine. Theor Prac Diag 2002; 4: 257-261.
13. Gao S, Tang CS. The cellular molecular mechanism of hyperhomocysteine in atherosclerosis. Dev Physiol 2002; 33: 335-338.
14. Yan H, Du JB, Tang CS. The regulatory role of the gasotransmitter hydrogen sulfide upon the aortic structural remodeling in spontaneous hypertensive rats. J Peking Univ (Health Sci) (Chin) 2004; 36: 234-237.
15. Kahleova R, Palyzova D, Zvara K, Zvarova J, Hrach K, Novakova I, et al. Essential hypertension in adolescents: Association with insulin resistance and with metabolism of homocysteine and vitamins. Am J Hypertens 2002; 15: 857-864. [PubMed]
16. Geng B, Chang L, Du JB, Tang CS. New strategies against homocysteinemia. J Peking Univ (Health Sci) (Chin) 2005; 37: 215-219.
17. Li X, Liu NK, Jiang HF, Pang YZ, Tang CS, Du JB. Homocysteine inhibiting the transfer of Arginine in human vascular endothelial cells. J Peking Univ (Health Sci) (Chin) 2002; 34: 159-162.
18. Sun XN, Li YM, Guo H. An approach on correlated factors of genetic polymorphism of the key enzyme in metabolism of homocysteine in patients with simple systolic hypertension. Chin Cardiovasc Dis J (Chin) 2003; 31: 269-273.
19. Yan H, Du JB, Tang CS. The alterations and meaning of the new gasotransmitter hydrogen sulfide in the pathogenesis of hypertension. Chin J Pediatr (Chin) 2004; 42: 172-175.
20. Yan H, Du JB, Tang CS. Effects of hydrogen sulfide on the thoracic aortic vasodilation in rats with spontaneous hypertension. Chin Pharmacol Bull (Chin) 2003; 19: 633-636.
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