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. Author manuscript; available in PMC: 2013 Feb 28.
Published in final edited form as: Obesity (Silver Spring). 2010 Dec 9;19(8):1560–1567. doi: 10.1038/oby.2010.294

Changes in Nitric Oxide, cGMP, and Nitrotyrosine Concentrations Over Skin Along the Meridians in Obese Subjects

Sheng-Xing Ma 1, Xi-Yan Li 1, Brian T Smith 1, Nainn-Tsyr Jou 1
PMCID: PMC3584154  NIHMSID: NIHMS445140  PMID: 21151015

Abstract

The purposes of these studies were to quantify the concentrations of total nitrate and nitrite (NOx−) cyclic guanosine monophosphate (cGMP), and nitrotyrosine over skin surface in normal weight healthy volunteers (n = 64) compared to overweight/obese subjects (n = 54). A semicircular plastic tube was taped to the skin along acupuncture points (acupoints), meridian line without acupoint (MWOP), and nonmeridian control and filled with a 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution for 20 min. The concentrations of NOx−, cGMP, and nitrotyrosine in the samples were quantified in a blinded fashion using chemiluminescence and enzyme-linked immunosorbent assay, respectively. In normal weight healthy volunteers, NOx− and cGMP concentrations were consistently increased over the pericardium meridian (PC) 4–7 compared with nonmeridian areas. NOx− concentration is enhanced over the bladder meridian (BL) 56–57, but cGMP level is similar between the regions. In overweight/obese subjects, NOx− contents were increased or tended to be elevated over PC and BL regions. cGMP is paradoxically decreased over PC acupoints and nonmeridian control on the forearm but the decreases were blunted along BL regions on the leg. Nitrotyrosine concentrations are markedly elevated (five- to sixfold) over both PC and BL in all areas of overweight/obese subjects. This is the first evidence showing that nitrotyrosine level is tremendously elevated over skin accompanied by paradoxical changes in nitric oxide (NO)-cGMP concentrations over PC skin region in overweight/obese subject. The results suggest that NO-related oxidant inflammation is systemically enhanced while cGMP generation is impaired over PC skin region but not over BL region in obesity.

Introduction

Overweight and obesity have increased dramatically and have become a major health problem around the world (1,2). The mechanisms of weight gain and obesity development are unclear. It has been suggested that inflammation might be a common denominator that links obesity to many of its pathologic sequels (3,4). Nitric oxide (NO) is a gaseous lipophilic free radical which has recently been found to have a key role in both normal physiological processes and in pathophysiological states such as chronic inflammation (5–7). NO stimulates guanylyl cyclase to generate cyclic guanosine monophosphate (cGMP), a second messenger directing vasodilatation and various biological functions including regulation of nutrition metabolism (8–10). Recent studies have demonstrated that an NO-cGMP signaling pathway mediates effects on lipolysis (10–13) and modifies the activity of key regulatory enzymes involved in fatty acid synthesis and oxidation (14). In another aspect, the presence of NO in biological systems also leads to the formation of oxidizing/nitrating reactive nitrogen species (ORNS) such as peroxynitrite (ONOO−) (6,7). Peroxynitrite is considered to be one of the major ORNS resulting from NO generation, and is a highly reactive oxidant contributing to inhibition of cGMP generation (15,16). Peroxynitrite has a very short half-life at neutral pH and reacts avidly with tyrosine residues in proteins to form nitrotyrosine, a stable end-product that is readily detectable, and can thus be used to monitor changes in ONOO−generation. Quantitation of nitrotyrosine has been used as a biomarker of ORNS production in various disease states such as chronic inflammation (17,18).

Acupuncture points (acupoints), as described in traditional Chinese medicine, are located along the meridian pathways (jingluo) and acupoints/meridians have been used in many unconventional medical practices such as acupuncture, moxibustion (heating acupoints), electrical stimulation, Qi Gong, and meditation (19,20). The meridian systems are believed to be physical pathway systems that deal with physiological regulation and pathological changes of the human body (19–21). Morphological studies have revealed that blood vessels, hair follicles, and nervous components are elevated in acupoints (22,23). Our previous studies have revealed that concentrations of total nitrite (NO2−) plus NO3−(NOx−) are consistently increased in three meridian skin regions associated with increased neuronal nitric oxide synthase protein levels in normal weight Sprague Dawley rats (24). The chemical labile nature of NO has been attributed to a rapid oxidation to both nitrite (NO2−) and nitrate (NO3−) (25,26), and measurements of these two stable metabolites (NO2− and NO3−) has been shown to be indicative of the concentration of NO in the tissue (25–27). In the living system, oxyhemoglobin and oxymyoglobin catalyze the complete conversion of NO or nitrite ion (NO2−) to nitrate ion (NO3−) (25,28). Recent studies have developed a new NO-scavenging compound, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), which has been used to absorb NO in biological systems (29,30). We have developed a painless, noninvasive device to collect NO from skin surfaces of acupoints and meridians using PTIO. Our recent results, using this biocapture device, have demonstrate that total nitrate plus nitrite (NOx−) concentrations are consistently increased over the pericardium meridian (PC) collected along the skin surface containing four acupoints, and the bladder meridian (BL) with two acupoints compared to nonmeridian control in humans (31).

The purpose of this study is to capture and quantify both NOx− and cGMP concentrations along the skin surface of acupoints compared to their corresponding meridian line without acupoint (MWOP) and nonmeridians controls in humans. NO and cGMP generations along the target areas of the skin surface were collected by a solution containing NO-scavenging compounds, PTIO, along the PC and BL. Nitrotyrosine concentrations were also measured simultaneously along the skin surface of acupoints compared to MWOP and nonmeridians controls in humans. The influence of overweight/obesity on the NO, cGMP, and nitrotyrosine productions over acupoints, MWOP, and nonmeridian area were examined in normal weight healthy volunteers compared to overweight/obese subjects.

Methods and Procedures

Subjects

A total of 118 men and women (18- to 65-years-old) recruited at Harbor-UCLA Medical Center volunteered for the following studies. Some subjects participated in more than one protocol in a randomized and blind fashion. All subjects were healthy, normotensive, and nonsmokers who did not have neither major surgery in the past 12 months nor history of cardiovascular disease. Subjects with dermatological problems, allergic disease, vascular disorders, infectious diseases, and prescribed medication were excluded from the study. The protocol was approved by the John F. Wolf, Human Subjects Committee of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. They were given a detailed oral instruction of the study and their informed consent was obtained. Female participants were not in their menstrual period on the study day. Experiments were performed in a quiet, air-conditioned room with the temperature maintained at 25–27 °C. All subjects were instructed to maintain their regular diet of breakfast or lunch, but not to eat or drink 2 h prior to the test. Researchers and technicians of the chemiluminescence Nitrogen Oxide Analyzer or performing the cGMP and nitrotyrosine measurements, and analyzing the data obtained were blinded to treatments of the subjects.

Determination of acupoints and meridians

The PC and the BL were studied in each subject. The locations of acupoints and meridians were identified by an acupoint/meridian map of human (19). As shown in Figure 1, PC and BL acupoints were defined as the meridian lines PC 4 to 7 containing four acupoints (Figure 1, top), and BL 56 to 57 with two acupoints (Figure 1, bottom). MWOP is defined as the distance between PC 3 and 4 and for BL as the distance between BL 55 and 56. Nonmeridian controls were obtained in nonmeridian areas close to PC or BL. These regions were chosen in the experiments by the following criterions: (i) acupoints/meridians in the regions can be easily identified on the body surface, (ii) there is enough distance away from other meridians to place the tube, and (iii) representations of acupoints/meridians on both the arm and leg.

Figure 1.

Figure 1

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Representative example of the biocapture device on skin surface. Pericardium meridian (PC or P, top left) and bladder meridian (BL or U.B., bottom left) in a human subject and related acupuncture points are illustrated (reproduced from Beijing College of Traditional Chinese Medicine et al., 1980). A small plastic tubing taped to the skin surface over the PC meridian and BL meridian lines. The region from PC 4 to 7 is defined as acupoint (four acupoints), the distance between PC 3 and 4 as meridian line without acupoint (MWOP), and nonmeridian control close to PC meridian (top). The area from BL 56 to 57 is defined as acupoint (two acupoints), the distance between BL 55 and 56 as MWOP, and nonmeridian control is defined over nonmeridian area adjacent to the meridian (bottom).

Instrumentation and NO collection protocol

Acupoints, MWOP, and nonmeridian control were defined as described previously in humans (31). Sterile distilled water was swabbed over the skin surface along the BLs and PCs and nonmeridian control areas two times at 5-min intervals. A biocapture device, which was developed by this lab, consists of a small plastic tube (0.5×7 cm) cut in half lengthwise and taped to the underside of the forearm or leg, as shown in Figure 1. Prior to the use the tube was immersed in 75% alcohol for 2 h and then dried in an incubator at 37 °C. On the study day, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 100 μmol/l) is dissolved in sterile normal saline and injected inside the sterilized tube that is attached to the surface of the skin and allowed to incubate for 20 min to absorb NO and other biochemicals. In a trial study, the optimal concentration of PTIO and the length of the incubation time required to absorb NO on the surface of the skin were determined before conducting the experiments. After the 20 min incubation, the liquid was collected from the tubes lying over acupoints, MWOP, and nonmeridian control area and transferred to a plastic vial for measurements of NOx− and cGMP concentrations. The tubing was then removed from the participant’s skin surface. The concentrations of NOx−, cGMP and nitrotyrosine in collected samples were then quantified in a blinded fashion (24,27,31,32).

Determination of NOx− concentrations

Concentrations of nitrite and nitrate (NOx−) were quantified using chemiluminescence as previously described (24,25,31). Measurements of NOx− concentrations from samples were conducted in a blinded manner. NOx− concentrations were measured with a Nitrogen Oxide Analyzer (SIEVERS, Boulder, CO). Briefly, samples are refluxed in the presence of 1.5 mmol/l vanadium (III) chloride in 2 mol/l HCl which quantitatively reduces both NO2− and NO3− to NO gas. This can then be quantified by chemiluminescence detection after reaction with ozone. The quantitative analysis was based on the standard curve established by measurements of peak areas of the standard NaNO2 compound. The lower limit of the detection of this assay was 0.1 pmol of NO.

Measurement of cGMP and nitrotyrosine concentrations

The concentrations of cGMP and nitrotyrosine in the collecting samples were assayed using a competitive enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) as described previously (17,18,32). The minimal detection level of nitrotyrosine and cGMP is 0.037 and 1.14 pmol/ml respectively. Briefly, 40 μl of the collected sample is diluted in 160 μl with Calibrator Diluent RD5P, and the diluted sample added to the 96 well microplate-coated with a goat anti-cGMP or anti-nitrotyrosine polyclonal antibody. Control (nonspecific binding) and standards are included in each assay. Horseradish peroxidaseconjugated rabbit polyclonal antibody to cGMP or nitrotyrosine are added to the diluted sample in the wells. After 3-h incubation at room temperature with 400 rpm shaking on an orbital microplate shaker, the wells were washed for four times to remove unbound cGMP/nitrotyrosine and primary antibody. The plate was developed by adding horseradish peroxidase substrate for 30 min at room temperature and stop solution was added to stop the reaction. The optical densities of yellow color development in the wells are read using a microplate reader set to 450 nm with a wavelength correction to 570 nm (Molecular Devices Emax, Sunnyvale, CA). Assays will be completed according to the manufacturer’s established protocols (17,18,32). Each assay will be standardized for intra- and interassay variability before use. The cGMP or nitrotyrosine concentration is calculated based on a standard curve in each assay.

Research protocols

NOx−, cGMP, and nitrotyrosine concentrations along the skin surface of acupoints, MWOP, and nonmeridian control in normal weight compared to overweight/obese humans. Volunteers (males and females, aged 18–55) were recruited with exclusion criteria including pregnancy, eating disorders, prescription medication, and serious medical condition. BMI was calculated by dividing the weight (in kg) by the squared height (in meters). BMI >25 is considered as overweight and over 30 as obese. Acupoints on skin surface of PC 4–7, BL 56–57, and their MWOP and nonmeridian controls were determined following the procedure described above (31,32). The biocapture device was attached to the skin surface over the PCs and BLs containing acupoints, MWOP (the meridian line between acupopints), and nonmeridian areas close to the meridians, as shown in Figure 1. PTIO solution (100 μmol/l) was placed inside the tubing and attached to the surface of the skin for 20 min. The liquid was drained from the tubing and kept in −80 °C. The concentrations of NOx− in the samples were quantified by using a chemiluminescence NO analyzer. cGMP and nitrotyrosine concentrations in the samples were quantified using enzyme-linked immunosorbent assay, respectively. The assays were conducted in a blinded fashion.

Chemicals

The drugs and chemicals used in these experiments were 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (Sigma), vanadium (III) chloride (Aldrich), and NaNO2 (Fisher).

Data analysis and statistical analysis

Results were expressed as mean ± s.e.m. of NOx− concentrations (nmol), cGMP and nitrotyrosine concentrations (pmol) per cm2 (length times width) of the collecting tube along the skin surface, respectively. The NOx−, cGMP and nitrotyrosine values were calculated by subtracting NOx−, cGMP and nitrotyrosine concentrations measured in 100 μmol/l PTIO from the total concentrations in each sample.

The significance of differences were determined by two factor-repeated analysis of variance, where the two factors are (i) three sites: acupoints, MWOP, and nonmeridian control and (ii) normal weight subjects compared to overweight/obese subjects. P values <0.05 were considered significant.

Results

Quantification of NO metabolites over skin surface of acupuncture points and meridians in normal weight compared to overweight/obesity

The study included 118 consecutive human subjects with 64 normal weight healthy volunteers (BMI < 25 kg/m2) and 54 overweight/obese subjects (BMI ≥ 25 kg/m2). The characteristics of the participants are detailed in Table 1. Body weight was significantly higher in overweight/obese subjects compared to normal weight subjects. The total population’s BMI was 21.4 ± 0.2 kg/m2 in normal weight healthy volunteers and 29.6 ± 0.6 kg/m2 in overweight/obese subjects. There was only a marginal significant increase in systolic and diastolic arterial blood pressure in overweight/obese subjects compared to normal weight subjects (P = 0.084). Compared to overweight/obese subjects, normal weight subjects were about 4 years younger, and more Asian subjects participated in this study.

Table 1.

Characteristics of participants

Characteristics Normal Weight (n = 64) Overweight/Obesity (n = 54)
Women, No. 34 28
Men, No. 30 26
Age, mean (SEM), year 34.4± 1.9 38.1± 1.5
Body Mass Index, mean (SEM) 21.4 ± 0.2 29.6 ± 0.6*
Weight, mean (SEM), pounds 130.3 ± 2.5 183.0 ± 4.7*
Systolic Arterial Pressure (mm Hg) 116.0 ± 2.7 128.7 ± 2.7
Diastolic Arterial Pressure (mm Hg) 66.4 ± 1.9 70.1 ± 3.6
White 20 13
Black or African American 9 11
Hispanic 8 13
Asian 24 11
More than One Race 2 4
Pacific Islander 1 4
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*

P < 0.05, compared to normal weight subjects.

Figure 1 (top panel) is a representative example of the location of acupoints, MWOP, and nonmeridian control along the PC on the forearm. As shown in Figure 1 (top panel), a semicircular plastic tube filled with PTIO was taped to the skin surface over acupoints PC 4–7, between PC 3 and 4 was defined as MWOP and for the nonmeridian control, the tubing was taped to the skin region adjacent to the defined PC acupoints. Figure 1 (bottom) is a representative example of the location of acupoints, MWOP, and nonmeridian control over the BL meridian on the leg.

Figure 2 shows NOx− concentrations over the skin surface of PC (left panel) and BL (right panel) acupoints, MWOP, and nonmeridian control in normal weight subjects compared to overweight/obese subjects. Our approach is based on using the biocapture device and PTIO solution in vivo to examine the NOx− concentrations along the skin surface of acupoints, MWOP, and nonmeridian regions. The sample consisted of 34 normal weight subjects (BMI = 21.4 ± 0.5 (mean ± s.e.)) and 31 overweight/obese (BMI = 30.0 ± 0.8). Analysis of variance of three sites showed that NOx− concentrations over PC and BL acupoints were significantly higher than those over MWOP and nonmeridian regions in normal weight healthy volunteers. However, such difference of NOx− levels between PC acupoints and nonacupoint areas did not exist in overweight/obese subjects, as shown in Figure 2 (left panel). NOx− concentrations over BL acupoints were still significantly higher than those over nonmeridian regions in overweight/obese subjects (Figure 2, right panel).

Figure 2.

Figure 2

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Quantification of nitric oxide (NO) metabolites over skin acupoints/meridians in normal weight compared to verweight/obese subjects. Concentrations of total nitrite plus nitrate (NOx−) along the pericardium meridian (PC, left) and bladder meridian (BL, right) in normal weight compared to verweight/obese subjects. NOx− concentrations (nmol/cm2) were obtained over PC 4–7 containing four acupoints compared to meridian line without acupoint (MWOP) and nonmeridian control (left panels). Right panels show that NOx− concentrations are collected from BL 56–57 over two acupoints compared to nonmeridian control. PC: the pericardium meridian; BL: the bladder meridian; without acupoint: nonacupoint areas along the meridians; control: nonmeridian control area adjacent to the meridians. Each bar represents the mean values and vertical bars represent s.e.m.

*P < 0.05, compared with nonmeridian control in normal weight subjects; #P < 0.05, compared with MWOP in normal weight subjects; +P < 0.05, compared with nonmeridian control in overweight/obese subjects.

Analysis of variance of the difference between overweight/obesity and normal weight showed that significant differences in NOx− concentrations (P < 0.05) were observed in the PC nonmeridian regions. In the BL meridian, NOx− concentrations were higher over nonmeridian regions in overweight/obese subjects, but the differences did not reach statistical significance (P = 0.208), as shown in Figure 2. In MWOP, overweight/obese subjects revealed marginally significant increases in NOx− concentrations compared to normal weight group in both PC (P = 0.138) and BL (P = 0.164). Compared to normal weight subjects with a higher level of NO content over acupoints, NOx− concentrations in overweight/obese subjects showed a slight elevation over PC acupoints, but did not attain statistical significance (Figure 2, left panel). NOx− concentrations over BL acupoints in overweight/obese suggested a moderate elevation compared to those collected on normal weight group. However, statistical analysis failed short of significance (P = 0.139) as shown in Figure 2 (right panel).

Analysis of cGMP concentrations over meridians/acupoints in normal weight compared to overweight/obesity

cGMP concentrations were examined in the samples collected along the skin surface of acupoints, and nonmeridian controls in the PCs and BLs. Figure 3 shows cGMP concentrations over acupoints, and nonmeridian control in normal weight healthy volunteers (BMI = 22.5 ± 0.4; n = 12) compared to a group of overweight/obese subjects (BMI = 30.9 ± 4.1; n = 10). In the PC on the forearm, cGMP concentrations were significantly increased over acupoints compared to nonmeridian controls, as shown in Figure 3 (left panels). cGMP levels over PC acupoints and nonmeridian control were significantly decreased in overweight/obesity compared to normal weight subjects. However, in BL meridian on the leg, cGMP concentrations over acupoints and nonmeridian control were similar. There were no detectable changes between normal weight and overweight/obese subjects over acupoints and nonmeridian control of BL meridian on the leg (Figure 3, right panel).

Figure 3.

Figure 3

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Quantification of cyclic guanosine monophosphate (cGMP) over skin acupoints/meridians in normal weight compared to overweight/obese subjects. Concentrations of cGMP along the pericardium meridian (PC, left) and bladder meridian (BL, right) in normal weight compared to overweight/obese subjects. cGMP concentrations (pmol/cm2) were obtained over PC and BL acupoints compared to nonmeridian control. Each bar represents the mean values and vertical bars represent s.e.m.

*P < 0.05, compared with nonmeridian control; #P < 0.05, compared to normal weight subjects. Other details are shown in Figure 2 legend.

Analysis of nitrotyrosine concentrations over meridians/acupoints in normal weight compared to overweight/obesity

The biocapture device was used to examine the levels of nitrotyrosine, a stable end-product of ONOO−, concentrations along the skin surface of acupoints, MWOP, and nonmeridian controls in healthy volunteers and a group of overweight/obese subjects. Figure 4 (top panels) shows nitrotyrosine concentrations over PC acupoints, MWOP, and nonmeridian controls on the forearm in normal weight healthy volunteers (BMI = 22.6 ± 0.6; n = 9) compared to a group of overweight/obese subjects (BMI = 28.5 ± 0.7; n = 10). Nitrotyrosine concentrations were significantly increased (sixfold more, P < 0.05) over PC acupoints, MWOP, and nonmeridan regions in overweight/obese subjects as compared to normal weight subjects (Figure 4, top). In normal weight subjects, nitrotyrosine concentrations over acupoints tended to be less compared to the same groups over MWOP and nonmeridian regions but the decrease failed short of statistical significance.

Figure 4.

Figure 4

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Quantification of nitrotyrosine over skin acupoints/meridians in normal weight compared to overweight/obese subjects. Concentrations of nitrotyrosine along the pericardium meridian (PC, top) and bladder meridian (BL, bottom) in normal weight compared to overweight/obese subjects. Nitrotyrosine concentrations (pmol/cm2) were obtained over acupoints, meridian lines without acupoint, and nonmeridian controls along the PC and BL meridian respectively. Each bar represents the mean values and vertical bars represent s.e.m. *P < 0.05, compared to normal weight subjects. Other details are shown in Figure 2 legend.

Figure 4 (bottom panel) shows that nitrotyrosine levels over BL acupoints, MWOP, and nonmeridian controls on the leg were significantly higher (four- to sixfold) in overweight/obese subjects (BMI = 32.6 ± 2.8; n = 7) compared to normal weight healthy volunteers (BMI = 22.1 ± 0.6; n = 11). Nitrotyrosine is present at very low levels over skin and there is no difference between acupoints compared to MWOP and nonmeridian area in normal weight healthy humans (Figure 4, bottom panels). These results suggest that ONOO− concentrations, as determined by nitrotyrosine levels, are markedly increased over skin surface in overweight/obese subjects as compared to normal weight subjects in both PC and BL areas.

Discussion

The goal of this study was to simultaneously collect and quantify the concentrations of NO and cGMP, and nitrotyrosine collected from skin surface over acupuncture points compared to MWOP and nonmeridian control areas along the PC on the forearm and the bladder (BL) meridian on the leg in humans. As well we investigated the effect of body weight (overweight/obesity) on NO and cGMP, and nitrotyrosine production over these same regions. The major new findings of this study are that: (i) in normal weight subjects, cGMP concentrations are consistently increased over the PC acupoints compared to nonmeridian area; (ii) in overweight/obese subjects, cGMP levels are decreased and NOx− concentrations tend to be increased over PC and nonmeridian areas; (iii) NOx− concentration is higher over BL acupoints compared with nonmeridian regions in normal weight subjects and enhanced in overweight subjects, but cGMP change is blunted in both normal and overweight subjects; and (iv) nitrotyrosine concentrations are five- to sixfold higher in overweight/obese subjects through all PC and BL acupoints, MWOP and nonmeridian areas. This is the first evidence showing that paradoxical changes in NO-cGMP concentrations exist over PC skin region on the forearm in overweight/obese subjects accompanied by a marked increase in nitrotyrosine levels. cGMP release is coupled with NO and is predominantly enhanced over PC acupoints in normal weight subjects. However, cGMP is uncoupled with enhanced NO generation and is paradoxically decreased over PC acupoints and nonmeridian control on the forearm in overweight/obesity. In BL meridian on the leg, cGMP levels did not significantly increase over acupoint in normal weight subjects and was not reduced in overweight/obese subjects as was observed in the PC on the forearm. Moreover, we unexpectedly found, using the same biocapture method, that nitrotyrosine concentrations are markedly increased in both PC and BL acupoints, MWOP and nonmeridian areas in overweight/obese subjects compared to normal weight subjects. The results suggest that NO-cGMP is physiologically released/generated from the skin surface with a high level at PC acupoints in normal weight subjects, and overweight/obesity exerts a marked increase ONOO− over skin accompanied by a decreased generation of cGMP on PC region. Clinically, use of the biocapture system on the PC, this may allow for a noninvasive way to monitor treatment that effect obesity and/or metabolic syndrome particularly where it pertains to the inflammatory aspect of NOergic species.

NO is a free radical produced by different isoforms of NOS from l-arginine and this reaction occurs in all mammalian tissues and cells, including adipocytes, brain, endothelial cells, heart, hepatocytes, macrophages, skeletal muscle, and skin tissue (33,34–38). NO activates soluble isoforms of guanylate cyclases, which generates cGMP, that serves as the second messenger of NO. The NO-cGMP pathway is an important signal transduction system, which affects various biological functions including nutrient metabolism (8–10). Recent studies have shown that dietary supplementation with l-arginine reduced fat mass and enhanced the expression of regulatory genes responsible for fatty acid oxidation and glucose metabolism in the Zucker diabetic fatty rat, an animal model of type 2 diabetes (37). Our previous studies have demonstrated that neuronal nitric oxide synthase-NO is enhanced in the skin and subcutaneous tissue of the acupoints/meridians in rats (24). Recent studies showed that NOx− concentrations were higher on the skin surface collected over acupoints compared to nonmeridian control areas (31). The present results show that cGMP production is parallel to NO levels, and NO-cGMP is produced at a higher level over PC acupoints in normal weight healthy humans. However, cGMP concentrations do not correspond to an increased NO contents but instead to decreased PC acupoints of overweight/obese subjects. Recent studies have suggested that multiple cGMP-dependent pathways are possibly involved in the underlying mechanisms of metabolic regulation (12,33). Several studies have reported that lipolysis, an early critical event in the utilization process of the stored triacylglycerol in adipose and various other tissues, is facilitated by natriuretic peptides through a cGMP-dependent signaling pathway (12,13,33). The lipolytic effect is mimicked by a nonhydrolyzabale analog of cGMP, 8-bromo-cGMP and promoted by protein kinase G, a downstream effector of cGMP (10,13). NO donors and cGMP analogs also inhibit in parallel fatty acid synthesis in particular inhibition of acetyl-CoA carboxylase activity, a key regulatory enzyme of fatty acid synthesis, in rat hepatocytes (14). cGMP plays an important role in lipid metabolism, and the current findings from this study suggest that abnormal release/generation of skin cGMP particularly over PC acupoints is involved in the pathological processes of overweight/obesity. Enhanced NO over skin acupoints and nonmeridian areas may reflect an inflammation process or a feedback increase in NO synthesis due to insufficient cGMP. Consistently, other investigators using a mouse model for metabolic syndrome have shown that ONOO− causes a decrease in cGMP production with an up-regulation of eNOS (39). These results also demonstrate that this biocapture method can be successfully used to monitor NO-cGMP and other chemicals released over acupoints along their meridian areas in vivo to monitor differences in physiological and/or pathological states.

It is well-documented that NO serves as a signaling molecule in physiological processes, but is also a significant source for ORNS such as ONOO− (5–7). Reactions converting NO into ONOO− can become predominant during oxidative stress (6,7). Several studies have demonstrated that NO can react in vivo with superoxide (O2−) to produce highly reactive ONOO− (17,18). A number of studies have reported that ONOO− is a potent oxidant and has been implicated in mediating vascular dysfunction and contributing to hypertension (4,40). While ONOO− has a very short half-life at neutral pH, stable end-product reactants such as nitrotyrosine are readily detectable. Quantitation of nitrotyrosine, which serves as a biomarker indicating presence of ORNS production, has been shown to be present in blood, urine, and other tissues and increased levels have been associated with various disease states such as hypertension and ischemia-reperfusion (15–18). Our studies reveal that nitrotyrosine can be quantified by enzyme-linked immunosorbent assay in the solution collected from skin surface using a novel biocapture device developed in this lab. The results also show that, compared to normal weight subjects, overweight subjects exhibit five- to sixfold higher nitrotyrosine levels uniformly over the skin surface (all acupoints, MWOP, and nonmeridian regions) but produced lower cGMP concentrations. Generation of ONOO− has also been shown to inhibit guanylate cyclase mediated generation of cGMP, and results in an impairment of normal lipolysis (10–13) and fatty acid metabolism (14). Our data support the possibilities and suggests that in the PC, enhanced ONOO− correlates to a reduction of cGMP generation and an increase in body weight. In all cases we observed increased levels of nitrotyrosine in overweight/obesity subjects as compared to control, but there is no direct correlation between nitrotyrosine concentration and BMI.

The environmental changes (such as global warming, and air pollutant inhalation) and excess food intake have been considered as factors of weight gain and obesity development. These factors have in fact been shown to promote systemic inflammation including oxidation of blood cells, a decreased reduced-oxidized glutathione, and increased ONOO− (4,41,42). The present studies clearly demonstrate that NOergic oxidant, ONOO−, is markedly increased in overweight/obese subjects, which distributes uniformly over the skin surface of the arm (PC) and leg (BL). At basal physiological condition in normal weight subject, ONOO− content is very low over skin surface and no detectable difference between acupoints and nonmeridian regions. The results provide direct evidence to support previous publications suggesting that systemic inflammation is involved in the pathophysiology of obesity. Whether elevation of the reactive oxidant and/or reduction of cGMP generation cause an impairment of normal lipolysis and/or fatty acid metabolism which contribute to obesity development is another important objective.

The mechanisms responsible for an increase in NO-cGMP concentrations over PC acupoints on the forearm in normal weight but paradoxical alteration in overweight/obesity are unclear. Previous studies from our lab showed that NO content and neuronal nitric oxide synthase expression are consistently higher in the skin acupoints/meridians associated with low electric resistance in rats (24). Recent studies have demonstrated that electroacupuncture stimulation increases the production of NO in arterioles (43). Electroacupuncture stimulation induces a significant release of dialysate NO and cGMP in the acupoint, but not in nonmeridian area in humans (32). As well, the activities of both neuronal and endothelial l-arginine-derived NO synthesis have been demonstrated in the skin tissue (8,22–24). Our results agree with these studies and further suggest that enhanced NO-cGMP concentrations over the acupoints may result from the rich distribution of blood vessels and neural fibers in the area. The present results support the previous report that activation of l-arginine-derived NO synthesis is high at acupoints and that elevation of NO-cGMP over the acupoints can be achieved through the activation of endothelial and/or neuronal NO synthesis/release system. In addition, the data also suggests that enhanced cGMP mediates the signaling functions of NO to lead a rich local microcirculation over acupoints/meridian in normal weight healthy humans. The results demonstrate that cGMP levels are decreased over PC and nonmeridian areas on the forearm in overweight subjects but not in BL meridian on the leg, which suggest that impairment of cGMP processes is meridian specific and/or location forearm/calf difference. The mechanisms of abnormal production of cGMP over skin surfaces particularly along PC region on the forearm in overweight/obesity require further investigation.

In summary, these results show, in normal weight healthy volunteers, that NO contents are consistently increased on skin surfaces of acupoints in the PCs and BLs compared to nonmeridian areas. cGMP release is coupled with NO and is predominantly enhanced over PC acupoints, but cGMP change is blunted on BL acupoints. In overweight/obese subjects, nitrotyrosine concentrations are increased five- to sixfold in both PC and BL acupoints, MWOP and nonmeridian areas. NO contents are significantly increased or tended to be elevated but cGMP concentrations were decreased overall acupoints, and nonmeridian areas compared to normal weight subject. These results suggest that in overweight/obese subjects there exist a marked elevation of ONOO−, a potent oxidant, over skin surface which is linked with an impaired cGMP generation over PC skin region on the forearm but not over BL region on the leg. The increase in the reactive oxidant, ONOO−, resulting from systemic inflammation is involved in the impairment of cGMP generation and the pathophysiological process of obesity, but the mechanisms/relationship of impaired cGMP generation over specific location on the forearm (PC) in obesity requires further study.

Acknowledgments

These studies were conducted at the biomedical research facilities of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center and NIH-supported General Clinical Study Center (GCRC) at Harbor-UCLA Medical Center. We are grateful to Dr. Peter Christenson, a Biostatistician from the GCRC, for statistical advice. This project was made possible by NIH grants (AT002478, AT004504, and AT004620) from the National Center for Complementary and Alternative Medicine, and a Research Award (ADA 7-07-RA-100) from the American Diabetes Association to S.-X.M.

Footnotes

Disclosure

The authors declared no conflict of interest.

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