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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:M137-M140 (2000)
© 2000 The Gerontological Society of America

Effect of Age and Menopause on Serum Concentrations of Pentosidine, an Advanced Glycation End Product

Masaaki Takahashia, Michio Oikawaa and Akira Naganoa

a Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Masaaki Takahashi, Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, 3600 Handa, Hamamatsu, 431-3192, Japan E-mail: taka1m{at}hama-med.ac.jp.

Decision Editor: William B. Ershler, MD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Background. Pentosidine is an advanced glycation end product. Our aim is to investigate (a) the age-related change of serum pentosidine and (b) the effect of menopause on serum pentosidine.

Methods. Using the high-performance liquid-chromatography method with column switching, we measured serum pentosidine in 140 healthy women aged 20–93 years. Serum creatinine was also measured. The samples of 13 young and 13 old subjects were used for the measurements of free pentosidine and fractions of pentosidine. Free pentosidine was measured without hydrolysis, and the fractions were measured with a 10,000 mol wt cutoff filter. To investigate the effect of menopause on pentosidine, two biochemical markers for bone turnover (CTx and osteocalcin) were measured in age-matched premenopausal and postmenopausal women (16 in each group).

Results. Serum pentosidine significantly increased with age (r = .702, p < .0001). The values of serum pentosidine for the groups beyond the age of 50 were significantly higher than those for the younger groups. The value for the group aged 80–93 years was three times higher than that for the group aged 20–29 years. Serum pentosidine moderately and significantly correlated to serum creatinine (r = .483, p = .0001). Free pentosidine was detected in only 3 of 13 young subjects and 2 of 13 old subjects. The ratio of free to total pentosidine was 2.9% and 1.2% in young and old subjects, respectively. Pentosidine <10,000 mol wt was not detected in all subjects. Pentosidine >10,000 mol wt was detected in all subjects. Serum CTx and osteocalcin significantly increased in postmenopausal women compared with those of premenopausal women. There was no significant change in serum pentosidine between the premenopause group and the postmenopause group.

Conclusion. Serum pentosidine significantly increased with age in healthy subjects aged 20–93 years and correlated to serum creatinine. The changes of fractions of pentosidine with aging were not observed. There was no effect of menopause on pentosidine.

PROTEINS in long-lived tissues are known to be modified when sugars are reduced posttranscriptionally. The early glycation products undergo a slow rearrangement to form irreversible advanced glycation end products (AGEs). AGEs play a major role in collagen's changes with aging, resulting in loss of elasticity, decreased proteolytic susceptibility, and accumulation of yellow and fluorescent substances. Although there are believed to be many kinds of AGEs, few are known. Pentosidine is one and is formed through the Maillard reaction (1). It has been reported to increase with age in various tissues (2)(3).

Pentosidine also has been reported to be significantly elevated in diseases such as diabetes mellitus, end-stage renal failure during hemodialysis, and rheumatoid arthritis (4)(5)(6)(7)(8)(9). These diseases and aging are known to be associated with osteopenia or osteoporosis. These facts suggest the hypothesis that pentosidine may directly or indirectly play a role on the pathogenesis of inducing osteoporosis. It indicates the possible involvement of AGEs in bone metabolism.

Although pentosidine has been reported to accumulate in tissues with age and has been implicated in increasing blood circulation with age, the age-related change of serum levels of pentosidine has not yet been elucidated in a significant number of subjects. It has been reported that pentosidine exists mainly in a protein-bound form in blood circulation and a free form of pentosidine exists as a few percentages of total pentosidine (10)(11)(12). Serum pentosidine binds mainly to high-molecular-weight proteins but not to low-molecular-weight proteins (13). We examined the change of form (fractions) of serum pentosidine with aging.

Pentosidine was previously found to be elevated in osteoporotic patients in a methodological report of measuring pentosidine in body fluids (12). Although the elevated levels of pentosidine in osteoporotic patients were suggested to have been caused by aging, because of the higher age of that group compared with that of the normal group, the involvement of AGEs in abnormalities of bone suggests that serum pentosidine reflects bone turnover. If this is true, serum pentosidine could increase after menopause because bone turnover increases after menopause, triggered by the dropping of estrogen levels in women. Therefore the aim of this study is to investigate (a) the age-related change of serum pentosidine and its fractions and (b) the effect of menopause on serum pentosidine.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Subjects
140 healthy women aged 20–93 years were involved in this study. All were volunteers and gave their informed consent before the study. Subjects who had present and past histories of diabetes mellitus, renal disease, liver disease, or any disease known to affect bone metabolism (such as hyperthyroidism, collagen disease, or ovarian tumor) were excluded from the study. The procedures followed were in accordance with the principles of the Declaration of Helsinki in 1975, as revised in 1983.

Methods
Measurement of serum pentosidine.-- 1 mL of serum sample was acid hydrolyzed with 1 mL of concentrated hydrochloric acid (HCl) and heated at 110°C for 20 hours in a sealed glass tube. The hydrolysate was filtered with a 0.45-µm membrane filter (DISMIC-25cs, Toyo Roshi Ltd., Tokyo, Japan); 125 µL of hydrolysate was mixed with 5 mL of water and evaporated under vacuum by a TC-8 concentrator (TAITEC, Tokyo, Japan). The residue was dissolved in 200 µL of 1% n-heptafluorobutyric acid (HFBA). The injection volume of the hydrolyzed serum sample, which was prepared as described above, was 20 µL. The method for measuring serum pentosidine is described elsewhere (12). Briefly, the high-performance liquid-chromatography (HPLC) system comprised a Model CCPM-II pump, which consisted of two pumps, a Model FS-8010 spectrofluorometer, a Model VC-8020 six-port switching valve, a Model SD-8022 air vacuum pump, a Model AS-8020 autosampler, and a Model SC-8020 supersystem controller (all from TOSOH, Tokyo, Japan). A gel-filtration column, TSK precolumn PW (4.6 mm x 3.5 cm) was used as column 1 (COL 1) and an ODS column, TSK-GEL ODS-80T (4.6 mm x 15 cm), was used as column 2 (COL 2) (two columns from TOSOH). The mobile phase (MP 1) for COL 1 was 5% acetonitrile containing 30 mmol/L of HFBA, and the mobile phase (MP 2) for COL 2 was 20% acetonitrile containing 30 mmol/L of HFBA. The flow rate was 1.0 mL/min. The detector (DET) was a spectrofluorometer used to detect internal fluorescence of pentosidine under the conditions of an emission at 385 nm and an excitation at 335 nm. At time zero, the sample was injected into COL 1, which was eluted with MP 1, and COL 2 was eluted with MP 2 at position A of a six-port switching valve. Just before the elution of pentosidine from COL 1 (the retention time being 2.8 minutes under this condition), the valve was switched to position B and the eluate fraction containing pentosidine was introduced into COL 2. After the elution of pentosidine (at a retention time of 4.2 minutes), the valve was switched back to position A. Then the introduced eluate was further separated by COL 2 and detected by the DET, and COL 1 was washed and conditioned with MP 2 until the next injection. The concentrations of pentosidine in serum were expressed in units of nanomoles per liter. Pentosidine was isolated from human articular cartilage. Methods of purification and characterization are described elsewhere (14).

Free pentosidine, fractions of pentosidine, and serum creatinine..-- Thirteen samples of young subjects (aged 23–40 years, mean 31.2 years) and 13 samples of old subjects (aged 71–88 years, mean 78.9 years) from this study's population were used for the measurements of free pentosidine and fractions of pentosidine. Free pentosidine was measured without hydrolysis of the serum sample (6). The serum (10 µl) was directly injected into the HPLC system. Serum (400 µl) was applied to a 10,000-mol wt centrifuge cutoff filter (Ultrafree, Millipore Corp., Bedford, MA) and centrifuged at 12,000 rpm, 8,060 g for 2 hours. The low- and the high-molecular-weight fractions (120 µl) were acid hydrolyzed and measured by HPLC, as described above. Serum creatinine was measured in all the subjects (N = 140) by a routine laboratory method.

Measurements of CTx and osteocalcin..-- To investigate the effect of menopause on serum pentosidine, two biochemical markers for bone turnover (CTx and osteocalcin) were measured in age-matched 16 premenopausal and 16 postmenopausal women. Menopausal status was ascertained, and women who had had no menstrual bleeding for at least 1 year were defined as being postmenopausal. Serum CTx (degradated product of type I collagen) was assayed by an enzyme-linked immunosorbent assay (ELISA) kit, Serum CrossLapsTM from Osteometer BioTec A/S (Herlev, Denmark) (15). The intra-assay and interassay coefficients of variation (CV) were 4.8% and 6.6%, respectively. Osteocalcin was measured by a two-site ELISA kit, an N-MIDTM osteocalcin ELISA from Osteometer BioTech A/S (Rodovre, Denmark). Two monoclonal antibodies that recognize midregion human osteocalcin (aa20-43) and N-terminus (aa7-19) were used in this assay (16). The intra-assay CV and interassay CV were 4.2% and 4.0%, respectively.

Statistical analysis..-- The statistical significance of difference was determined with Mann–Whitney U tests between two groups and with one-way analysis of variance (ANOVA) followed by the Scheffé F test among more than two groups. Simple regression was performed for univariate correlation, and the statistical significance of correlation was determined with the Spearman rank correlation test. The analyses were performed with StatView II (Abacus Concepts, Inc., Berkeley, CA) on a Macintosh computer (Apple Computer, Cupertino, CA); p < .05 was considered significant.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Serum pentosidine significantly increased with age (y = 3.59x - 19, n = 140, r = .702, p < .0001 by Spearman rank correlation test) (Fig. 1). The values of serum pentosidine for the groups beyond the age of 50 were significantly higher than those for the younger groups (Table 1 ). The value for the group aged 80–93 years was three times higher than that of the group aged 20–29 years. Serum pentosidine moderately and significantly correlated to serum creatinine (Pentosidine [nmol/l] = 269.7 x creatinine [mg/dl] - 67.2, r = 0.483, p = .0001).



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  		Figure 1. Age-related changes of serum pentosidine. y = 3.59x - 19, n = 140, r = .702, p < .0001 by Spearman rank correlation test.

 

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  		Table 1. Values of Serum Pentosidine for the Different Age Groups

 
Free pentosidine was detected in only 3 of 13 young subjects and 2 of 13 old subjects (Table 2 ). Mean concentration of serum free pentosidine of young subjects was 2.8 nmol/l and the ratio of free to total pentosidine was 2.9%. The mean concentration of serum free pentosidine of old subjects was 4.7 nmol/l and the ratio of free to total pentosidine was 1.2%. Pentosidine in the low-molecular-weight fractions (<10,000) was not detected in 13 young and 13 old subjects. Pentosidine in the high-molecular-weight fractions (>10,000) was detected in all subjects.


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  		Table 2. Free Pentosidine and Fractions of Pentosidine in Serum

 
To investigate whether serum pentosidine levels reflect bone turnover, the effect of menopause on serum pentosidine was examined. Sixteen premenopausal and 16 postmenopausal women were randomly selected for an age-matched comparison (Table 3 ). Serum CTx and osteocalcin significantly increase in postmenopausal women compared with those of premenopausal women (p < .001). There was no significant change in serum pentosidine between premenopause and postmenopause as determined by a Mann–Whitney U test ( p = .3657).


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  		Table 3. Effect of Menopause on Serum Pentosidine and Biochemical Markers for Bone Tumors

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
It has been reported that pentosidine accumulated with age in tissues such as dura mater (1), lens (6), skin (17), cartilage (14)(18), glomerular basement membrane (19), aorta (20), and meniscus (21). However, there have been few reports on the measurements of pentosidine in body fluids and on the age relationship of pentosidine therein. Although we have previously reported that the urinary excretion of pentosidine exponentially increased from children to elderly people (90 years old) (22), in a separate paper serum pentosidine was measured in a relatively small population and the range of age was limited to 60 years (10). In that study, serum pentosidine moderately but significantly increased with age (coefficient correlation, 0.453). The present study demonstrates serum pentosidine as increasing to the age of 93 years in a greater population with a broader range of age. The coefficient correlation was 0.702. The values of serum pentosidine for the groups beyond age 50 were significantly higher than those for the younger groups. The value for the group aged 80–93 years was threefold times that of the group aged 20–29 years.

Because AGEs are harmful products for the body, they are removed physiologically. However, an increase of serum pentosidine in elderly people indicates that all pentosidine cannot be removed by aging. In the present study, serum pentosidine moderately but significantly correlated to serum creatinine. Because pentosidine is mostly catabolized and eliminated through the kidney, the dysfunction of the kidney may play a major role in an increase of serum pentosidine with aging. However, the synthesis of pentosidine may increase with aging because oxidative stress increases with age and oxidation is involved in formation of pentosidine. Serum pentosidine levels reflect the degree of accumulation of pentosidine in the body. Therefore serum pentosidine can be a useful biochemical marker for evaluating the accumulation of AGEs and aging.

There is evidence that most of pentosidine exists as a protein-bound form in blood circulation but most of pentosidine exists as free form in urine (12). It was reported that free pentosidine was detected in patients undergoing hemodialysis in whom serum pentosidine greatly elevated; however, free pentosidine was not detected in normal subjects because the free form of pentosidine exists as only a small percentage of the total pentosidine in serum. In this study, free pentosidine was detected in only 3 of 13 young subjects and 2 of 13 old subjects and the free to total ratio of pentosidine was 1.2%–2.9%. Those results were in accordance with the previous studies. Friedlander and colleagues reported that in patients with peritoneal dialysis or hemodialysis, 95% of the pentosidine was bound to proteins >10,000 mol wt, <1% of the pentosidine to proteins <10,000 mol wt, and <1% of the pentosidine as free (13). In their data, the pentosidine bound to proteins <10,000 was less than free pentosidine (approximately 1/3 of free). In the present study, pentosidine in the low-molecular-weight fractions was not detected in all young and old subjects. The current results demonstrate that most of pentosidine in normal subjects is bound to proteins >10,000 mol wt and the great changes of fractions of pentosidine with aging were not observed. However, it is necessary to validate the relationship between fractions of serum pentosidine.

The formation of AGEs in the extracellular matrix is implicated in the development of chronic diabetic complications. Abnormalities in bone metabolism such as osteopenia and delayed fracture healing are often seen in diabetic patients. AGEs inhibit osteoblastic cell differentiation and function in vitro, and they significantly increase in the cortical bone of rats because of diabetes and advanced age (23)(24). AGE-modified proteins stimulate monocyte/macrophage to secrete bone-resorbing cytokines such as IL-1ß, IL-6, and TNF-{alpha} (25). When unfractioned mouse bone cells containing osteoclasts were cultured on dentin slices, AGE-modified proteins increased the number of resorption pits formed by osteoclasts (26). AGEs enhanced the production of the bone resorption factor IL-6 in human bone-derived cells and increased the levels of IL-6 mRNA in the cells (27). Therefore AGEs may play a role in the development of osteopenia. In women after menopause, it is well accepted that bone turnover increases, triggered by the dropping of estrogen levels. The biochemical markers of bone turnover have been proven to be elevated in postmenopause to reflect the increased bone turnover. CTx (degradated product of type I collagen) and osteocalcin have been extensively used as biochemical markers for bone metabolism. In the present study, to investigate the possibility of pentosidine as a bone marker, the effect of menopause was examined on serum pentosidine compared with CTx and osteocalcin. Contrary to our expectation, serum pentosidine did not change after menopause whereas the markers for bone turnover did increase. However, further research (such as a longitudinal study) is needed to examine the possibility of pentosidine for reflecting bone metabolism.


   Acknowledgments
 
We thank Mika Matsumoto for her technical assistance.

Received March 17, 1999

Accepted July 29, 1999


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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