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Technical Briefs |
Departments of
1
Clinical Pathology and
2
Pediatrics, College of Medicine, Inha University Hospital, Inchon, 400-103, South Korea
3
Department of Pathology, Nagoya University Hospital, Nagoya, 466-0065 Japan
4
Department of Pharmacology and Toxicology, Medicinal Toxicology Center, College of Medicine, Inha University, Inchon, 400-103, South Korea
aaddress correspondence to this author at: Department of Clinical Pathology, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Inchon, 400-103, South Korea; fax 82-32-890-2529, e-mail jwchoi{at}inha.ac.kr
NO is produced in many different cells and is involved in the regulation of such physiological and pathophysiological processes as inflammation, vasodilation, and metabolism (1). Depending on the cell type, NO is produced in an enzymatic reaction catalyzed by one of the three isoforms of NO synthase (NOS): neuronal NOS, endothelial NOS, and inducible NOS (2). Measurement of the NO metabolites, nitrate and nitrite (NOx), is also important as a marker of NOS enzyme activity.
Obesity is a condition involving an excess accumulation of body fat, and the prevalence of obesity is rapidly increasing worldwide. Excessive weight and obesity are leading to nutrition-related disorders of clinical and public health concern. Recent studies have suggested a role for NO in the regulation of food intake in an animal experiment (3)(4). Endothelial and inducible NOS have been shown to be present in adipose tissue of the rat (5), suggesting that adipose tissue may be a potential source of NO production. Previous reports demonstrated that NOS activity and inducible NOS protein were also present in human subcutaneous adipose tissue (6) and showed that inhibition of NOS led to increased lipolysis in this tissue (7). However, how NO production changes as body mass index (BMI) increases in apparently healthy subjects has not been studied extensively. Moreover, correlation studies of serum NOx concentrations, body fat mass, and blood lipid concentrations in healthy subjects are limited. Therefore, in the present study, we investigated the changes in NOx concentrations according to BMI and evaluated the relationships among NOx concentrations, total body fat, and lipid profiles in adolescents.
We measured serum NOx concentrations, complete blood cell counts, iron
markers, and serum lipid profiles in 185 males and 178 females, 14–19
years of age, selected from 1473 students attending middle or high
school. Eight anthropometric measurements were taken in all subjects:
weight; height; circumference of upper chest, upper arm, waist, and
hip; and subscapular and triceps skinfold thickness measured by a
caliper. Body adiposity was assessed using a leg-to-leg bioelectric
impedance device, the TANITA body fat analyzer (TBF-611; Tanita), which
enables simultaneous measurements of body weight, impedance, fat-free
mass, total body water, and fat percentage in a subject standing on the
stainless steel electrode (8). BMI was determined as weight
in kilograms divided by the square of the height in meters
(kg/m2). The reference interval of BMI is defined
as 19.0–24.9 kg/m2, overweight as a BMI of
25–29.9 kg/m2, and obesity as a BMI >30
kg/m2 (9). The males and females were
divided into three groups according to BMI: overweight group (BMI
25.0 kg/m2), healthy weight group (19.0 
BMI < 25.0 kg/m2), and underweight group
(BMI <19.0 kg/m2). We compared the data in
extremely lean adolescents (BMI <16.0 kg/m2)
with those in obese adolescents (BMI 30.0
kg/m2). We excluded 24 subjects in this study—12
adolescents who showed evidence of chronic or recent infections, 7 who
had histories of iron or vitamin supplementation, and 5 who had
previously undergone surgical operations—because they had
inflammation-induced increases in NOx. This study was approved by the
Ethical Committee at Inha University Hospital. The subjects were given
a detailed description of the study before their consent was obtained.
Venous blood (7 mL) was drawn into iron-free evacuated serum separator tubes after 12 h of fasting. NOx concentrations were measured by a NADPH-dependent nitrate reductase assay (10) in the serum of adolescents subjected to a reduced nitrate and nitrite diet. After the serum nitrate (NO3-) was converted to nitrite (NO2-) by NADPH-dependent nitrate reductase (incubated with glucose-6-phosphate dehydrogenase and NADPH in 14 mmol/L sodium phosphate buffer, pH 7.4), the total concentration of nitrite was determined spectrophotometrically at 540 nm. We measured NOx directly in the sera without deproteinization and without dilution because according to our results for 35 specimens selected randomly from the subjects, there were no significant differences in NOx concentrations between deproteinized and nondeproteinized sera or between diluted and nondiluted sera. To avoid diet effect on serum NOx concentrations, the subjects were given a list of foods potentially rich in nitrate and were requested to abstain from these foods before sample collection after an overnight fast. Specifically, cured meat, fish, cheese, herbal or black teas, beer, wine, and malted beverages were excluded from the diet (11).
Complete blood cell counts, serum iron markers and lipid profiles, and serum ferritin were assayed with a SE 9000 electronic counter (Sysmex), a Hitachi 747 automatic chemical analyzer (Hitachi), and an ACS:180 chemiluminescence assay (Chiron), respectively. Data analysis was conducted using the SAS 6.12 software package (SAS Institute). To compare the differences of values, we used the Kruskal–Wallis test before Mann–Whitney comparisons were made between groups. Correlation coefficients were calculated by the Spearman method. P <0.01 was considered statistically significant.
The changes in serum NOx concentrations, iron markers, and lipid
profiles according to BMI are available as a data supplement at
Clinical Chemistry Online
(http://www.clinchem.org/content/vol47/issue6). The
NOx concentrations in the underweight subjects did not
differ significantly from those in subjects in the healthy weight
group. However, the serum NOx concentrations were 4.1- and 4.2-fold
higher in overweight male and female subjects (BMI 25.0
kg/m2), respectively, than in the underweight
subjects (BMI <19.0 kg/m2). On the other hand,
serum NOx concentrations changed in parallel with the anthropometric
variables. In this study, there were no significant differences in body
weight, body fat, and skinfold thickness between underweight and
healthy weight subjects; however, overweight subjects showed large
differences in anthropometric variables when compared with underweight
adolescents. In particular, mean body fat was 1.5-fold higher in girls
than in boys, whereas mean NOx concentrations were 1.3-fold higher in
girls than in boys. Moreover, the NOx concentrations were 13.9-fold
higher in the obese subjects (BMI 30.0 kg/m2)
than for the extremely lean subjects (BMI <16.0
kg/m2). These results indicate that NO production
increases in obese human and that this increase begins from the time
that BMI is >25 kg/m2 in both males and females.
Because iron deficiency may up-regulate NOS activity (12),
in this study we measured iron markers to strictly compare NOx
concentrations between extremely lean and obese subjects. Markedly
increased NOx concentrations in obese subjects (BMI 30.0
kg/m2), who showed no significant differences in iron
markers from the extremely lean adolescents, suggest that NO
biosynthesis increases overtly in obese humans irrespective of iron
status. Moreover, as shown in Fig. 1
, NOx concentrations correlated positively with the obesity
(r2 = 0.207; P <0.01) and
BMI (r2 = 0.211; P <0.01),
and with the body fat (r2 = 0.259;
P <0.01) in the overweight females (BMI 25.0
kg/m2). Our results are in accordance with a
previous report showing that NOS activity is present in human adipose
tissue and produces NO through inducible NOS from a source of NO,
adipose tissue per se (7). On the other hand, results
contradictory to ours have also been reported. Ferlito and Gallina
(13) demonstrated that in diabetics, being overweight,
increased blood pressure, and diabetes mellitus per se cause a
nonsignificant increase of NO production in comparison with healthy
controls. Andersson et al. (14) found that plasma nitrate
concentrations were not different between obese and control women.
However, Ferlito and Gallina (13) measured plasma nitrite in
patients with type 1 and 2 diabetes, and Andersson et al.
(14) measured plasma nitrate in obese and nonobese
postmenopausal women. In our study, we measured NOx in apparently
healthy adolescents 14–19 years of age without any diseases. We
therefore believe that the discrepancies of the results seem to be
derived from the differences in subjects among the studies.
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To investigate how BMI and body fat change with serum NOx
concentrations, we compared data according to NOx concentrations. There
were no significant differences in anthropometric variables, iron
markers, and lipid profiles between the subjects with NOx <20 µmol/L
and with NOx 20 µmol/L. However, the mean values of BMI, weight,
and body fat showed significant differences when we compared the
subjects with NOx <20 µmol/L to the subjects with NOx 80 µmol/L.
In particular, body fat and skinfold thickness were 1.6-fold higher in
the subjects with NOx 80 µmol/L than in the subjects with NOx <20
µmol/L. These results indicate that moderately increased NOx
concentrations are relevant to BMI and body fat. In this study, we
investigated whether serum lipid profiles show significant changes as
NOx concentrations increase; however, unlike body fat mass, there were
no significant differences in serum lipid profiles between the subjects
with NOx <20 µmol/L and with NOx 80 µmol/L.
In conclusion, our results suggest that obesity leads to increased NO production in humans. Increased serum NOx correlate strongly with body fat but poorly with serum lipid concentrations.
Acknowledgments
This work was supported by a Science Research Center grant from Korea Science and Engineering Foundation to the Nitric Oxide Radical Toxicology Research Center (NORTRC).
References
The following articles in journals at HighWire Press have cited this article:
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  N. Mehebik, A.-M. Jaubert, D. Sabourault, Y. Giudicelli, and C. Ribiere Leptin-induced nitric oxide production in white adipocytes is mediated through PKA and MAP kinase activation Am J Physiol Cell Physiol, August 1, 2005; 289(2): C379 - C387. [Abstract] [Full Text] [PDF]
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 H. Yan, E. Aziz, G. Shillabeer, A. Wong, D. Shanghavi, A. Kermouni, M. Abdel-Hafez, and D. C. W. Lau Nitric oxide promotes differentiation of rat white preadipocytes in culture J. Lipid Res., December 1, 2002; 43(12): 2123 - 2129. [Abstract] [Full Text] [PDF]
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