Serum Homocysteine, B12 and Folic Acid ... - Hogrefe eContent

Int. J. Vitam. Nutr. Res., 73 (1), 2003, 8–14

Serum Homocysteine, B12 and Folic Acid Concentration in Thai Overweight and Obese Subjects Rungsunn Tungtrongchitr1, Praneet Pongpaew1, Chuthaporn Tongboonchoo1, Niyomsri Vudhivai1, Supranee Changbumrung1, Anchalee Tungtrongchitr2, Benjaluck Phonrat3, Duangkamol Viroonudomphol1, Somchai Pooudong1 and Frank Peter Schelp4 1

Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University 2 Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University 3 Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University 4 Institute of International Health, Center for Humanity and Health Sciences, Free University, Berlin and Humboldt University of Berlin, Germany

Received for publication: July 5, 2002

Abstract: This study investigated levels of serum homocysteine, vitamin B12, folic acid, vitamin B6 and vitamin C, in 37 male and 112 female overweight and obese Thai volunteers (body mass index; BMI ≥ 25.00), and 23 male and 90 female normal-weight Thai volunteers, who came for a physical check-up at the Out-patient Department, General Practice Section, Rajvithi Hospital, Bangkok from March to October of 2000. Data included anthropometric measurements and waist/hip ratios. All anthropometric variables, except height, were significantly higher for the overweight subjects than for the normal subjects. Statistically significantly higher levels of serum homocysteine were found in the overweight subjects. Serum homocysteine concentrations in overweight and obese males were significantly higher than in overweight and obese females. Serum folic acid and vitamin C in the overweight and obese were found to be statistically significantly lower than in the control subjects. No statistically significant difference in vitamin B12 was found in the overweight and obese subjects compared with the normal control subjects. The medians of serum folic acid and vitamin C concentrations for the overweight and obese males were significantly lower than those of the overweight and obese females. A negative correlation was found between serum folic acid and homocysteine concentrations in all overweight and obese subjects. A significant negative correlation between serum folic acid and vitamin B6 was observed in both male and female overweight and obese subjects. The results of the investigation suggest that homocysteine levels in overweight and obese subjects seem to be caused by insufficient dietary folic acid intake and probably not by B12 deficiency. Key words: Homocysteine, B12, folic acid, obese

Int. J. Vitam. Nutr. Res., 73 (1), 2003, © Hogrefe & Huber Publishers

R. Tungtrongchitr et al: Homocysteine, B12, Folic Acid, Obese

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obese people compared with apparently healthy subjects in Bangkok.

Introduction Obesity, a health problem reaching epidemic proportions in western countries, is an important risk factor for the most frequently occurring diseases in the western world, i.e. diabetes, hypertension, certain types of cancer, and cardiovascular diseases [1]. Rapid economic development in the Asia-Pacific region has brought about a population move from a less developed to an industrialized society, with an associated change from a low-calorie diet to the high availability of palatable foods and the risk of increasing weight gain. In Thailand, the transition from a rural to an urban environment affects health in various ways via changes in eating habits, alcohol consumption, and smoking [2–4]. The rapid approach towards a status of a newly industrialized country is clearly reflected in some demographic and economic indicators [5] of Thailand. A total of 23.6% Thai female construction site workers were reported to be obese [6]. Obesity is found in 11% of the Thai elderly [7]. Moderate to severe obesity is increasingly found. This might be associated with clear health risks, including hypertension, diabetes, and dyslipidemia; leading to a higher risk of cardiovascular disease [8]. McCully and Wilson [9] proposed the “homocysteine theory of arteriosclerosis” in 1975, based upon pathological examinations of autopsy material from children with hyperhomocysteinuria [10]. Moreover, the results from about 100 clinical and epidemiological studies have shown that even a mild and moderate elevation of homocysteine can be associated with an increased risk of cardiovascular diseases [11]. Homocysteine is formed from the essential amino acid methionine as a product of numerous S-adenosylmethionine-dependent transmethylation reactions [12]. Homocysteine may be remethylated to methionine. This reaction is in most tissues catalyzed by the ubiquitous enzyme 5-methyltetrahydrofolate-homocysteine-S-methyltransferase (methionine synthase), which requires vitamin B12 as a cofactor and methyltetrahydrofolate as a cosubstrate [12]. Alternatively, homocysteine is degraded to cysteine via the transsulfuration pathway in two sequential vitamin B6-dependent reactions [13]. Knowledge and information on the homocysteine and vitamin status of the Thai overweight and obese is needed to correct shortcomings, improve quality of life, reduce risk of chronic diseases, especially cardiovascular diseases, and increase lifespan. However, information on serum homocysteine and the vitamin status of the overweight and obese is limited, especially in developing countries such as Thailand. This study was conducted to investigate homocysteine and vitamin status as well as anthropometric measurements of healthy overweight and

Materials and Methods Study Population The study population comprised 37 male and 112 female overweight and obese Thai volunteers, including 23 male and 90 female normal-weight subjects. Thai volunteers who attended the Out-patient Department, General Practice Section, Rajvithi Hospital, Bangkok, for a physical check-up, were investigated for this study. All of them visited the clinic voluntarily. Except for minor ailments and typical diseases of obese people such as hypertension, mild to moderate degree of cardiovascular diseases, and non-insulin dependent diabetes mellitus, these persons were still fairly healthy, as diagnosed through physical and biochemical laboratory examinations for the inclusion criteria. Age, marital status, place of origin, drinking, and smoking habits were assessed by standardized questionnaires. The same medical doctor conducted the physical examinations throughout the study.

Analytical Methods The body weight of each individual dressed in light clothing was measured using a carefully calibrated beam balance (Detecto®, Detecto Scale Manufacturing, USA). Height measurements were taken using a vertical-measuring rod. The body mass index (BMI) or Quetelet’s index was conventionally calculated as weight in kg/ (height in meters2). The classifications of BMI employed were those used by the WHO Expert Committee, 1995 [14], overweight grade I: BMI = 25.00–29.99 kg/m2; grade II (obese): BMI = 30.00–39.99 kg/m2; grade III (obese): BMI ≥ 40 kg/m2. Waist and hip circumferences were also measured to calculate waist/hip ratio (normal value for female < 0.77, male < 0.90) [15–16]. From the subjects under study, about 10 mL of venous blood were taken in the morning after an overnight fast. Red blood cell hemolysate was prepared and stored at –20°C for not longer than one week and used afterwards for vitamin B6 determinations. The active form of vitamin B6 is a very important coenzyme in many biological mechanisms. In particular, the activity of the erythrocyte enzymes, aspartate aminotransferase (EAST), and its activation coefficients (AC) after stimulation with the respective coenzymes are sensitive indicators of the nutritional status of pyridoxine, respectively [17]. Vitamin B6 status was assessed using erythrocyte aspartate amino-


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transferase activity [18]. Values of 2.00 α EAST and above indicate a vitamin B6 deficiency status. Serum vitamin C was determined according to the method described by Liu et al [19]. Values below 5 µg/L indicate a deficiency in vitamin C. Serum vitamin B12 and folic acid were assessed using a commercially available radio-immunoassay Dual-count solid-phase no-boil assay for vitamin B12/folic acid [Diagnostic Products Corporation (DPC), Los Angeles]. Values indicating deficiencies are serum vitamin B12 < 200 pg/mL; serum folic acid < 3.0 ng/L [20]. Serum homocysteine was measured by a fluorescence polarization immunoassay (Homocysteine; Abbott) on an automated analyzer (IMx system; Abbott). Optimal procedures in blood sample collection and handling were followed to prevent the passage of homocysteine from red cells to serum and thus ensure reliable measurements. The cutoff point separating normal from abnormally increased serum concentrations was 15.0 µmol/L, which is in agreement with the literature [21].

Statistical Analysis The results were expressed as median, range, and 95% confidence interval (CI). The data were coded and analyzed using a standard statistical method provided by the Minitab computer program [22].

Results Median, range, and 95% confidence interval (CI) of age, anthropometric variables, folic acid, vitamins B12, B6, and C, and homocysteine in overweight and control subjects are shown in Table I. All the anthropometric variables, except height, were significantly higher for the overweight subjects than the normal subjects. There were statistically significantly higher levels of serum homocysteine observed in the overweight than in the control subjects. Meanwhile, serum folic acid and vitamin C in the overweight and obese were found to be statistically significantly lower than in the control subjects. The median

Table I: Medians, ranges, and 95% confidence interval (CI) of age, anthropometric variable, blood pressure, vitamin status, and homocysteine in overweight and control subjects Parameter

Total Overweight (N = 149) Median 95%CI (range)

Age (yrs) Weight (kg) Height (m) BMI (kg/m2) Waist (cm) Hip (cm) Waist/Hip Ratio Folic acid (ng/L) Vitamin B12 (pg/mL) Vitamin B6 (αEAST) Vitamin C (mg/L) Homocysteine (mol/L

40.0 (18.0–60.0) 76.0 (54.0–153.0) 1.57 (1.45–1.86) 31.50 (25.10–56.20) 91.0 (66.5–127.0) 108.0 (86.5–151.5) 0.84 (0.67–1.01) 5.00 (1.70–19.00) 500.0 (120.0–1600.0) 1.40 (0.80–2.26) 3.50 (0.00–21.00) 9.98 (5.71–22.86

38.0–41.0 74.5–78.5 1.56–1.58 30.60–32.30 89.6–93.0 106.0–110.0 0.83–0.85 4.40–5.30 430.0–560.0 1.33–1.46 3.00–4.69 9.53–10.53

BMI = body mass index α EAST = erythrocyte aspartate aminotransferase activity coefficient * Mann-Whitney U-Wilcoxon Rank Sum W Test (Two-Tailed) Int. J. Vitam. Nutr. Res., 73 (1), 2003, © Hogrefe & Huber Publishers

P-value* Control (N = 113) Median (range)

95%CI

38.0 (18.0–60.0) 54.2 (42.5–78.0) 1.58 (1.43–1.85) 21.80 (18.50–25.00) 72.5 (60.0–98.0) 92.0 (82.0–102.0) 0.78 (0.65–0.93) 10.50 (1.90–24.50) 580.0 (100.0–1850.0) 1.62 (1.01–3.40) 6.50 (0.00–23.00) 9.19 (2.65–16.61)

36.0–40.0

0.195

52.8–55.9

0.000

1.56–1.59

0.222

21.19–22.20

0.000

71.4–74.0

0.000

92.0–94.0

0.000

0.77–0.80

0.000

9.93–11.50

0.000

500.0–643.7

0.104

1.52–1.73

0.000

5.50–7.69

0.000

8.73–9.66

0.005

Table II: Medians, ranges, and 95%(C.I.) of age, anthropometric variable, blood pressure, vitamin status, and homocysteine in overweight and control subjects between male and female Parameter

Male Overweight (N = 37) Median 95%CI (range)

Age (yrs)

34.2–45.9 81.2–91.1 1.67–1.72 28.99–31.28 93.0–103.0 103.4–110.0 0.91–0.94 4.20–5.57 371.0–580.0 1.27–1.59 1.50–3.47 12.17–14.47

Female Overweight Control (N = 112) (N = 90) Median 95%CI Median 95%CI (range) (range)

P-value*

39.0 (19.0–58.0) 62.0 (50.7–78.0) 1.67 (1.57–1.85) 22.21 (18.70–24.40) 80.5 (64.0–89.0) 94.0 (87.0–102.0) 0.87 (0.72–093) 8.50 (3.50–17.50) 630.0 (185.0–1250.0) 1.74 (1.02–2.24) 5.00 (0.00–12.50) 11.96 (7.49–16.61)

30.0–42.0

0.132

0.504

60.0–64.5

0.000

1.65–1.70

0.645

20.62–23.16

0.000

74.7–83.7

0.000

91.0–95.4

0.000

0.82–0.88

0.000

7.63–10.28

0.000

439.0–685.5

0.264

1.61–1.95

0.000

3.22–6.11

0.006

10.17–12.39

0.035

38.5 36.0–42.0 37.5 34.2–40.0 (18.0–60.0) (18.0–52.0) 74.2 72.8–75.7 52.5 51.8–54.0 (54.0–153.0) (42.5–78.0) 1.55 1.55–1.56 1.56 1.55–1.57 (1.45–1.70) (1.43–1.78) 30.86 30.27–31.61 21.90 21.45–22.35 (25.10–56.20) (18.47–24.97) 89.8 86.0–91.0 71.0 70.0–72.4 (71.0–127.0) (60.0–98.0) 108.0 106.0–110.0 92.0 91.6–93.2 (86.5–151.5) (82.0–111.0) 0.83 0.81–0.84 0.77 0.76–0.78 (0.70–0.99) (0.65–0.88) 5.30 4.63–5.59 11.00 10.50–12.00 (1.70–15.00) (1.01–3.40) 505.0 430.0–568.5 580.0 500.0–640.0 (120.0–1600.0) (100.0–1850.0) 1.40 1.32–1.46 1.56 1.49–1.71 (0.80–2.26) (1.01–3.40) 4.50 3.20–5.80 6.25 6.00–8.38 (0.00–21.00) (0.00–23.00) 9.58 8.90–9.98 8.89 8.29–9.56 (5.71–22.86) (2.65–14.50)

BMI = body mass index α EAST = erythrocyte aspartate aminotransferase activity coefficient * Mann-Whitney U-Wilcoxon Rank Sum W test (Two-Tailed)

0.000 0.222 0.000 0.000 0.000 0.000 0.000 0.211 0.020 0.000 0.038

R. Tungtrongchitr et al: Homocysteine, B12, Folic Acid, Obese 11


42.0 (18.0–60.0) Weight (kg) 85.0 (62.4–127.0) Height (m) 1.69 (1.51–1.86) BMI (kg/m2) 30.49 (25.10–47.90) Waist (cm) 98.5 (66.5–122.0) Hip (cm) 98.5 (89.5–129.0) Waist/Hip Ratio 0.92 (0.67–1.01) Folic acid (ng/L) 4.60 (1.70–19.00) Vitamin B12 (pg/mL) 490.0 (120.0–1550.0) Vitamin B6 (αEAST) 1.45 (1.02–1.90) Vitamin C (mg/L) 2.50 (0.00–18.50) Homocysteine (mol/L 13.01 (8.08–18.55)

P-value* Control (N = 23) Median 95%CI (range)

12


serum folic acid concentration in the overweight subjects was 5.00 (range 1.70–19.00 ng/L) compared with 10.50 (range 1.90–24.50 ng/mL) in the control subjects (p < 0.001). The medians of serum folic acid and vitamin C concentrations in overweight and obese males were significantly lower than those in overweight and obese females (Table II). The serum homocysteine concentrations in overweight and obese males were significantly higher than in overweight and obese females. A negative correlation was found between serum folic acid and homocysteine concentrations in both sexes of overweight and obese subjects (r = –0.301, p < 0.05). Figure 1 shows the relationship between serum homocysteine and folic acid concentration to be seemingly linear (Y = 12.18–0.288 X). A significant negative correlation between serum folic acid and vitamin B6 was observed in both male and female overweight and obese subjects (r = –0.205, p < 0.05). The regression relationship of serum vitamin B6 and serum folic acid was shown in Figure 2 (Y = 1.532–0.019 X).

Discussion This investigation shows that statistically significantly higher levels of serum homocysteine were observed in the overweight subjects than in the control subjects. By using > 15 µmol/L of serum homocysteine as the cutoff point, 9.4% (14 out of 149) of overweight and obese subjects were found to have elevated homocysteine levels, compared with only 1.8% (2 out of 113) of control subjects.

Homocysteine ( µmol/L) 30

20

Y =12.18 - 0.288X

10

0 0

10

20

Folic acid (ng/L)

Figure 1: Homocysteine versus folic acid concentration in obese Thai.

Vitamin B6 (αEAST) 2.4 2.2 2.0

Y = 1.532 - 0.019 X

1.8 1.6 1.4 1.2 1.0 .8 .6 0

10

20

Folic acid (ng/L)

Figure 2: Vitamin B6 versus folic acid concentration in obese Thai.

Hyperhomocysteinemia was found predominantly in the male overweight and obese subjects (27%, 10 out of 37) compared with 3.6% (4 out of 112) in the females. Hyperhomocysteinemia has been recognized as an important independent cardiovascular risk factor [23]. It is hypothesized that homocysteine alters endothelial and smooth muscle cell function by generating reactive oxygen species [24–26]. In the authors' previous report, it was also found that the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase, which are responsible for metabolizing reactive oxygen species (ROS), were significantly lower in the obese groups than in the normal groups [27]. This results in oxidative stress elevation and diminishes antioxidative capacity, which increases the risk of atherosclerotic vessel disease in these subjects [28–29]. Serum folic acid levels in the overweight and obese subjects were found to be statistically significantly lower than in the control subjects. However, no statistically significant differences in vitamin B12 levels were found in the overweight and obese subjects compared with the normalweight control subjects. Folic acid and vitamin B12 play a role in the transfer of methyl groups and their presence therefore is important for DNA and RNA synthesis and amino acid metabolism. Homocysteine derives from the essential amino acid methionine. It may be retroconverted to methionine through vitamin B12 and folic acid-dependent pathways, or degraded via a vitamin B6-dependent route [29]. Therefore, hyperhomocysteinemia in overweight and obese subjects seems to be caused by insufficient dietary folic acid intakes and probably not through B12 deficiency.



The median values of vitamin C concentration for both sexes of overweight and obese subjects were found to be lower than for the control subjects (Table I). Serum vitamin C concentrations in males were also found to be lower than in female overweight and obese subjects (Table II). More than 50% (92 out of 149) of all overweight and obese subjects had vitamin C levels below the normal cutoff point value of 5 µg/L. The low levels of vitamin C concentrations in overweight and obese subjects might result from high activity metabolism and might be related to decreased oxidative enzymes that require a reducing agent such as vitamin C to maintain the situation (?). Entry of homocysteine into the transsulfuration pathway is catalyzed by B6-dependent cystathionine β-synthetase, which has limited tissue distribution [12]. B6-dependent cystathionine β-synthetase deficiency will lead to hyperhomocysteinemia [30–33]. Although the high rate of vitamin B6 deficiency in Thailand is well known, as confirmed by recent studies undertaken in elderly [34], road sweepers [35], construction site workers [36], vegetarians [37], newborns and their mothers from the northeast [38], children and their mothers in well-baby clinic [17], and preschool children in the northeast of Thailand [18], no statistically significant differences in vitamin B6 were found when comparing overweight and obese subjects with normal subjects (Table I). The same result was found when the data were analyzed for separate sexes (Table II). Furthermore, there seemed to be a higher percentage of B6 deficiency in the controls than in the overweight and obese subjects (19.5% in controls, 2.7% in overweight and obese subjects). Therefore, hyperhomocysteinemia in overweight and obese subjects might not result from vitamin B6 deficiency.

Acknowledgements The authors wish to express their sincere thanks to all our volunteers, Dr. Ariya Lertchavanakul, Outpatient General Practice Section, Rajvithi Hospital, and to all staff of the Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University for their cooperation in this research. This work was partly supported by funds from Mahidol University and the Free University, Berlin, Germany.

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Prof. Frank Peter Schelp Institute of International Health Center for Humanity and Health Sciences Free University, Berlin and Humboldt University of Berlin Berlin, Germany Tel./Fax +49 30 8445 1281/1280
