Effect of Age on Bone Mineral Density and the Serum Concentration of Endogenous Nitric Oxide Synthase Inhibitors in Rats

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Rong Lu, PhD,1 Chang-Ping Hu, PhD,1 Xian-Ping Wu, MD,2 Er-Yuan Liao, MD,2 and Yuan-Jian Li, MD1,*

Results of previous studies have indicated that bone mineral density (BMD) is decreased in aged animals and elderly humans, and that treatment with nitric oxide (NO) donors prevents bone loss. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, can inhibit NO synthesis. In the study reported here, we examined age-related changes in the serum content of ADMA and in BMD in various skeletal regions. The BMD in the lumbar part of the spine, the femur, and the tibia in 12-month-old rats was markedly increased, compared with that in 6-month-old rats, and the BMD in 20-month-old rats was decreased, compared with that in 12-month- old rats. Serum concentration of ADMA in 20-month-old rats was significantly increased, compared with that in 6- or 12-month-old rats. A similar age-related change in the concentration of lipid peroxide also was seen in the three age groups. These results suggest that the increased amount of endogenous ADMA may be associated with an age- related decrease in BMD in rats.

Osteoporosis has been defined as a disease characterized by normal adult bone, but is largely expressed in response to in- decreased bone mineral density (BMD), leading to enhanced flammatory stimuli (10, 11). An L-arginine analogue, such as bone fragility and consequent in risk of fracture. Epidemiologic asymmetric dimethylarginine (ADMA), which is widely present investigations have indicated that the incidence of osteoporosis in the blood of humans and animals (12), can inhibit NOS activ- in elderly men is far higher than that in young men. However, ity and decrease NO synthesis in vivo and in vitro (13, 14). Re- the mechanisms responsible for age-related osteoporosis are not cent clinical investigation has indicated that an increase of fully understood. Multiple factors, such as calcitonin, parathyroid serum ADMA concentration is positive correlation with advanc- hormone, and estrogen, have been found to be important regula- ing age (15). We hypothesize that the age-related changes of tors of bone cell function. Results of recent investigations have BMD may be associated with age-related change in ADMA con- indicated that nitric oxide (NO) has important effects on bone tent. The mechanisms responsible for the increase of ADMA functions (1-3). It has been reported that the decrease of NO syn- content in aging rats are unknown. Previous studies have indi- thesis contributes to the development of osteoporosis in vivo and cated that the increased concentration of ADMA is related to in vitro and that NO donors, such nitroglycerin and nitrates, in- the increase in peroxides, such as malondialdehyde (MDA), in crease BMD in osteoporotic animals and humans (4, 5). Others hypercholesterolemic rabbits (16, 17). Therefore, in the study have reported that long-term administration of L-nitroarginine reported here, we examined the effect of age on BMD and the methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, serum content of ADMA and MDA in the rats. causes a decrease in BMD in the femur and the lumbar part of the spine in clinically normal and ovarietomized female rats (6, Materials and Methods 7). However, the decreased BMD can be reversed by administra- Animals. Healthy, male, Sprague-Dawley rats aged four, 10, tion of L-arginine or NO donor nitroglycerin (4, 5, 8). Further- and 18 months (n = 16 in each age group), were purchased from more, results of clinical studies have indicated that nitrates Laboratory Animal Center of Shanghai (Shanghai, P.R. China) enhance BMD in people with osteoporosis (9). and were raised for two months in our laboratory animal faculty. Nitric oxide is produced by NOS catalyzing L-arginine degra- The rearing environment in the two laboratories was similar: dation. At least three NOS isoforms, including endothelial NOS room temperature, relative humidity, ventilation, and lighting (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), have were 23 ± 1∞C, 55 ± 5%, 15 air changes/h, and 12 h' light/12 h' been identified in bone tissues (3, 10, 11). The eNOS is widely dark, respectively. All animals were kept under the same condi- expressed on a constitutive basis in various bone cells, whereas tions and were supplied with food and water ad libitum. The nNOS is mainly expressed in bone lining cells and in osteocytes study was carried out according to the guidelines set by the Cen- (10). The iNOS does not appear to be expressed constitutively in tral South University Veterinary Medicine Animal Care and Use Committee. Received: 12/11/01. Revision requested: 1/24/02. Accepted: 3/15/02. Mean body weight was 480 ± 15, 608 ± 20 and 630 ± 16 g for 1Department of Pharmacology, 2Institute of Metabolism and Endocrinology, six-month-old (young adult), 12-month-old (adult), and 20- Xiang-Ya School of Medicine, Central South University, Changsha, Hunan 410078, People’s Republic of China. month-old (senescence) rats, respectively. Before surgical proce- *Corresponding author. dures, rats were anesthetized by intraperitoneal administration

224 Effect of age on bone density and nitric oxide synthase in rats

AB C

Figure 1. Analyzing images of rat lumbar part of the spine (A) and femur (B) and tibia (C) in vitro by use of dual energy x-ray absorptiometry. of pentobarbital (60 mg/kg of body weight). After blood for bioas- Table 1. Effect of age on bone mineral density (BMD [g/cm2]) in the lumbar say was collected from the carotid artery, samples from the fe- part of the spine of rats murs, the tibias, and the lumbar spine from 4th to 6th were Age resected. The bones were placed in sterile saline and stored at -20∞C 6 mon 12 mon 20 mon until examination. R-1 0.1671 ± 0.0041 0.2349 ± 0.0021* 0.2172 ± 0.0037† Measurement of BMD. Bone mineral density was measured R-2 0.1695 ± 0.0030 0.2490 ± 0.0036* 0.2108 ± 0.0039† by use of dual energy x-ray absorptionetry (DXA) with a Hologic R-3 0.1764 ± 0.0041 0.2677 ± 0.0027* 0.2211 ± 0.0029† Total 0.1731 ± 0.0036 0.2510 ± 0.0029* 0.2180 ± 0.0033† QDR 4500 A fan beam x-ray bone depsitometer (Hologic Inc., Waltham, Mass.), which has been thought to be a precise and ef- *P < 0.01 vs 6-month value; †P < 0.01 vs 12-month value. R-1 = a sixth of the lumbar part of the spine; R-2 = a fifth of the lumbar part fective method of measuring BMD (18, 19). The preparations of the spine; R-3 = a fourth of the lumbar part of the spine. were positioned supine on the table and were orientated perpen- Total = average BMD of R1–3. dicular to the pass of the x-ray beam. They were scanned on Values are mean ± SEM, n = 16. perspex in an ultrahigh resolution mode, a scanstep of 0.254 mm, and the scan speed was 6.077 mm/s. Seven equal lines of the drofuran (22:77:1 [vol:vol:vol) at a flow rate of 1 ml/min. whole length of the femur and the tibia, and the lumbar part of The thiobarbituric acid reaction substance inflecting lipid per- the spine 4 to 6 were analyzed (Fig. 1). The BMD was expressed oxide was examined by use of a spectrofluorometer set at emis- as grams per square centimeter. sion wavelength of 515 nm and excitation wavelength of 553 nm, Determination of the serum content of ADMA and and was expressed as the content of malondialdehyde (MDA). malondialdehyde. The blood samples were centrifuged at 1,300 Reagents. Standard asymmetric dimethylarginine (ADMA) (¥g for 15 min (4∞C), and the serum was de-proteinated by addi- and 5–SSA were purchased (Sigma Chemical Co.). tion of 5-sulfosalicylic acid (5-SSA, Sigma Chemical Co., St Louis, Statistics. Results are expressed as mean ± SEM. The values Mo.). The precipitated protein was removed by centrifugation at were determined by use of analysis of variance and the Newman- 2,500 (¥g for 20 min (4∞C), and the supernatant was used for Keuls Student t test. A value of P < 0.05 was accepted as significant. measurement of ADMA, using high-performance liquid chromatography (HPLC) as described (20), with some modification. The Results HPLC was carried out, using a Shimadzu LC-6A liquid chro- Bone mineral density. The BMD value in the lumbar part of matograph with Shmadzu SCL-6A system controller and the spine at 12 months of age was higher than that at 6 months, Shimadzu SIC-6A autosampler (Kyoto, Japan). o-Phthaldia- whereas the BMD at 20 months of age was less than that at 12 dehyde adducts of methylated amino acids and internal standard months (Table 1). Similarly, an age-related alteration in BMD of ADMA (Sigma Chemical Co.) produced by pre-column mixing the femur and the tibia was seen (Tables 2 and 3). were monitored, using a model RF 530 fluorescence detector set Serum content of ADMA and MDA. There were no signifi- at lex = 338 and lem = 425 nm on a Resolve C18 column. Samples cant differences in serum concentration of ADMA at 6 and 12 were eluted from the column, using a linear gradient containing months of age (P > 0.05), but the serum value of ADMA at 20 mobile phase A composed of 0.05M (pH 6.8) sodium acetate- months of age was markedly increased, compared with that at 6 methanol-tetrahydrofuran (81:18:1 [vol:vol:vol]) and mobile and 12 months (Fig. 2A). A similar alteration in MDA content phase B composed of 0.05M sodium acetate-methanol-tetrahy- was seen in the three age groups (Fig. 2B).

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Table. 2. Effect of age on BMD (g/cm2) in the femur of rats

Lines Left femur Right femur 6 mon 12 mon 20 mon 6 mon 12 mon 20 mon

R-1 0.2750 ± 0.0129 0.3265 ± 0.0057* 0.2964 ± 0.0029† 0.2685 ± 0.0032 0.3243 ± 0.0044* 0.2863 ± 0.0021† R-2 0.1823 ± 0.0034 0.2118 ± 0.0044* 0.1844 ± 0.0018† 0.1764 ± 0.0039 0.2049 ± 0.0040* 0.1804 ± 0.0016† R-3 0.1552 ± 0.0020 0.2334 ± 0.0019* 0.2025 ± 0.0035† 0.1449 ± 0.0024 0.2259 ± 0.0015* 0.1983 ± 0.0043† R-4 0.1748 ± 0.0024 0.2716 ± 0.0024* 0.2238 ± 0.0034† 0.1665 ± 0.0024 0.2571 ± 0.0010* 0.2225 ± 0.0047† R-5 0.2001 ± 0.0022 0.2938 ± 0.0022* 0.2431 ± 0.0047† 0.1902 ± 0.0031 0.2744 ± 0.0015* 0.2385 ± 0.0046† R-6 0.2264 ± 0.0028 0.2985 ± 0.0025* 0.2452 ± 0.0038† 0.2156 ± 0.0030 0.2856 ± 0.0023* 0.2394 ± 0.0036† R-7 0.1997 ± 0.0028 0.2544 ± 0.0024* 0.2241 ± 0.0018† 0.1879 ± 0.0033 0.2485 ± 0.0024* 0.2184 ± 0.0016† Total 0.2044 ± 0.0024 0.2712 ± 0.0020* 0.2335 ± 0.0021† 0.1949 ± 0.0027 0.2613 ± 0.0014* 0.2287 ± 0.0027† Total = average BMD of R1–7. See Table 1 for key.

Table 3. Effect of age on BMD (g/cm2) in the tibia of rats Lines Left tibia Right tibia 6 mon 12 mon 20 mon 6 mon 12 mon 20 mon

R-1 0.2322 ± 0.0034 0.2864 ± 0.0038* 0.2431 ± 0.0023† 0.2278 ± 0.0037 0.2748 ± 0.0040* 0.2402 ± 0.00822† R-2 0.1558 ± 0.0020 0.2074 ± 0.0016* 0.1905 ± 0.0021† 0.1485 ± 0.0025 0.2016 ± 0.0019 * 0.1840 ± 0.0018† R-3 0.1528 ± 0.0017 0.1953 ± 0.0019* 0.1886 ± 0.0022† 0.1465 ± 0.0021 0.1989 ± 0.0024* 0.1787 ± 0.0023† R-4 0.1501 ± 0.0022 0.1961 ± 0.0021* 0.1847 ± 0.0022† 0.1419 ± 0.0023 0.1993 ± 0.0028* 0.1738 ± 0.0024† R-5 0.1538 ± 0.0027 0.2084 ± 0.0036* 0.1846 ± 0.0022† 0.1485 ± 0.0021 0.2027 ± 0.0021* 0.1751 ± 0.0023† R-6 0.1320 ± 0.0022 0.2205 ± 0.0035* 0.1806 ± 0.0030† 0.1568 ± 0.0028 0.2102 ± 0.0018* 0.1745 ± 0.0025† R-7 0.1759 ± 0.0025 0.2269 ± 0.0024* 0.1928 ± 0.0026† 0.1700 ± 0.0024 0.2192 ± 0.0080* 0.1985 ± 0.0023† Total 0.1747 ± 0.0019 0.2252 ± 0.0021* 0.1987 ± 0.0021† 0.1692 ± 0.0022 0.2193 ± 0.0018* 0.1936 ± 0.0019† See Tables 1 and 2 for key. Discussion Age-related changes in BMD have been documented in humans and animals (19, 21). An increase in BMD appears with age from immaturity to young adult, and BMD reaches the peak value to a certain adult stage. However, this change is not uni- form for all tissues in all regions. For example, the peak value is earliest at 20 to 24 years of age in the trochanter and Ward’s regions of femur, and latest at 40 to 44 years of age in the distal third of the forearm in humans (21). However, BMD decreases with advancing age in senescent individuals (22). In the study reported here, the BMD in the lumbar part of the spine, the femora, and the tibia was significantly increased in 12-month-old rats, compared with young adult rats (6 months), whereas the BMD in these tissues was decreased in 20-month-old, compared with 12-month-old rats. It has been suggested that decreased NO synthesis may facilitate development of osteoporosis. In vitro and in vivo studies have indicated that constitutive production of NO (derived from eNOS pathway) is essential for normal bone cell functions (23, 24). In cultured osteoblast cells, the role of estrogen in osteoblast cells was correlated with activation of NO/eNOS-dependent signaling pathways (25, 26). In eNOS gene-deficient mice, osteoblast maturation and activity are defective, postnatal bone formation is markedly retarded, and the anabolic effects of estrogen on bone formation are blunted (2, 27, 28). Other studies have indicated that the activity of eNOS decreases in postmenopausal women and elderly men as well as aged animals (29, 30). These findings indicate that the eNOS isoform has a key role in regulation of bone cell functions, and decreased eNOS activity may be an important factor contributing to the development of age-related osteoporosis. Decreased BMD also has been associated with some pathologic conditions, such as rheumatoid arthritis, an inflammatory joint disease. There is evidence to suggest that the iNOS pathway is involved in mediation of cytokine- or inflammation-induced bone Figure 2. Concentrations of asymmetric dimethylarginine (ADMA loss. For example, inflammation-induced osteoporosis was medi- [A]) and malondialdihyde (MDA [B]) for three age groups of rats. **P < 0.01 vs 6-month value; ++P < 0.01 vs 12-month value. Values are ated in part by activation of the iNOS pathway and was reversed mean ± SEM, n =16. 226 Effect of age on bone density and nitric oxide synthase in rats

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