- Email: [email protected]
Urinary excretion of deoxypyridinoline in 24-hour and first-void samples in healthy Turkish children
Clinical Biochemistry, Vol. 33, No. 4, 269 –272, 2000 Copyright © 2000 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/00/$–see front matter
PII S0009-9120(00)00072-2
Urinary Excretion of Deoxypyridinoline in 24-Hour and First-Void Samples in Healthy Turkish Children HANIFI SOYLU,1 S¸U¨KRU¨ ARAS,3 N. ONUR KUTLU1 , MU¨CAHIT EGˇRI,2 and KAMIL S¸AHIN4 Departments of 1Pediatrics and 2 Public Health, I˙no¨nu¨ University, Turgut O¨zal Medical Center, Malatya, Turkey; 3Department of Biochemistry, Vakıf Gureba Teaching Hospital, Istanbul, Turkey; 4 Department of Pediatrics, Anadolu Hospital, Istanbul, Turkey Objectives: Collagen cross-links are formed during the maturation process of bone matrix. They have been considered as valuable markers in some metabolic, endocrinologic, and neoplastic bone disorders. As an advantage, it can be measured in urine as well as in serum samples. However, the excretion characteristics remains controversial. Design and methods: We investigated urinary free deoxypyridinoline (f-Dpd) excretion in first-void urine samples and in 24-hour collections in healthy Turkish children. We also evaluated the possible correlations and gender-related differences in Dpd excretion between these sampling methods. Both urine samples of 62 subjects (aged from 31 to 120 months) were analyzed by Immulite chemiluminescent technique. Results: There were no remarkable differences in f-Dpd excretion between first-void and 24-hour urine samples, although f-Dpd values of the first-void samples were slightly higher (Dpd: creatinine, mean ⫾ SD, 20.5 ⫾ 5.8 nmol/mmol vs. 19.6 ⫾ 5.6 nmol/mmol, respectively, p ⬎ 0.05). A strong linear correlation was found between 24-hour and first-void urine f-Dpd excretions (r ⫽ 0.77, p ⬍ 0.05). In addition, f-Dpd showed no gender-related differences between boys and girls in either 24-hour or first-void urine samples (p ⬎ 0.05). Conclusions: Because of difficulties in long-time urine collection in infants and young children, f-Dpd assessment in first-void single urine samples is an easy, safe, and non-invasive method. Copyright © 2000 The Canadian Society of Clinical Chemists
KEY WORDS: deoxypyridinoline; childhood; first-void single urine.
(Dpd) occur and stabilize mature collagen chain within the extracellular matrix (2). The cross-links are formed during the maturation, not the biosynthesis, of collagen and release into circulation and excreted by urine without metabolization (3– 6). This is a continuous process in normal growth and bone remodeling (3– 6). Some markers for bone metabolism such as osteocalcin, bone specific alkaline phosphatase, and propeptides that derived from type I collagen, have disadvantages such as requirement of serum assessment, whereas Pyd and Dpd can be measured in either serum or urine (7). Some authors had preferred single urine assessment in their studies, however, the others collected 24-hour one (7,8). The single urine assessment is easier and more practical. Twenty-four hour urine collection is troublesome and often requires hospitalization especially in infants and young children. However, there is only a few comparative data between these methods in childhood (7,9). To clarify this situation, we aimed to compare free Dpd (f-Dpd) levels between urine samples of first-void and 24-hour in order to determine whether the first-void urine has any value in determination of f-Dpd excretion.
Introduction he organic matrix of bone is a complex-organized structure that undergoes mineralization. It is largely the product of the osteoblasts, and consisted mainly of type I collagen, which has functional cross-linked propeptides that are located to the telopeptide region (1). Their lysyl or hydroxylysyl components are converted by lysyl hydroxylase or lysyl oxidase to aldehydes during the maturation. Thus, pyridinoline (Pyd) and deoxypyridinoline
T
Correspondence: Dr. Hanifi Soylu, I˙no¨nu¨ University, ¨ zal Medical Center, Department of Pediatrics, Turgut O Malatya, Turkey. E-mail: [email protected]. Manuscript received December 22, 1999; revised February 29, 2000; accepted March 23, 2000. CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
Material and methods SUBJECTS Sixty-two healthy Turkish children were enrolled in this prospective study. By history, clinical examination, and routine laboratory assessment, none of the subjects had any evidence of bone, hepatic, renal, or endocrine diseases or malnutrition, and none of them was taking medication that acts on bone metabolism. The parents were informed about the aim and method of the study. Age (months), height (cm), and weight (kg) of each child were recorded. No diet or exercise restriction was done prior to the study. 269
SOYLU
URINE
ET AL.
COLLECTION AND ANALYTICAL METHOD
Two different samples were obtained, one from a 24-hour sample and the other from the first morning void urine sample. The parents were educated about how the urine collection should be performed. The urine bags were used for collection of the samples when needed in young children. The urine sample was centrifuged, and the supernatant was stored without preservative at ⫺20 °C until the analysis. Deoxypyridinoline measurement was performed by chemiluminescent enzyme immunoassay (CEI) according to Immulite method (Immulite Pyrilinks-D, Catalog No:LKPD1 Diagnostic Products Corporation, CA, USA). This test is a solid phase containing a polystyrene bead that is coated with monoclonal antibody specific for Dpd. At the study phase, the urine specimen and alkaline phosphatase-conjugated Dpd were simultaneously introduced into the test unit and incubated for 30 min at 37 °C with intermittent agitation for competition of the sample with the enzymelabeled Dpd. Unbound enzyme conjugate was then removed by a centrifugal wash with distilled water, afterwards concentration was measured by the luminometer. Test results were normalized to the urinary creatinine concentration to correct for variations in urinary flow. Urinary creatinine was assessed by BM CREA creatinine kit (Boehringer-Mannheim, Germany, Hi Co Creatinine, Catalog No: 1040847) in Hitachi 717 biochemical analytical system (Boehringer-Mannheim Hitachi, Germany-Japan). Each urine sample was diluted as a ratio of 1/10. Each Dpd result (nmol/L) was divided by the sample’s creatinine (Cr) value (mmol/L), therefore, the results were expressed as nmol Dpd/mmol Cr. STATISTICAL
METHODS
The SPSS computerized statistical software (SPSS, version 7.5.1, Statistical Package for the Social Sciences Inc., Chicago, IL, USA) was used for the statistical analyses. Differences between mean Dpd values of both urinary samples and differences between both sexes were determined by Student’s t test. The correlation between 24-hour and first-void urine samples was assessed by Pearson correlation test. Statistical significance was ascribed when p was equal to or less than 0.05.
Figure 1 — Urinary f-Dpd excretion in 24-hour and the first-void urine samples in girls and boys (p ⬎ 0.05).
Results The study population consisted of 62 children (35 boys, 27 girls) aged 31 to 120 months (mean age ⫾ SD; 69.88 ⫾ 25.65 months). There were no statistically significant differences between ages and weights of both sexes, except heights. The heights of boys were significantly taller than girls (119.5 ⫾ 11.9 cm vs. 108 ⫾ 18.2 cm, in respect, p ⬍ 0.05). Urinary Dpd levels were similar between both genders in the first-void urine (boys; 19.4 ⫾ 5.8 nmol Dpd/mmol Cr vs. girls; 21.9 ⫾ 5.4 nmol Dpd/mmol Cr, p ⬎ 0.05) and in the 24-hour urine samples (boys; 18.6 ⫾ 5.3 nmol Dpd/mmol Cr vs. girls; 20.9 ⫾ 5.4 nmol Dpd/mmol Cr, p ⬎ 0.05) (Figure 1). The physical characteristics of study population and statistical features were shown on Table 1. We did not observe any significant difference in Dpd values between 24-hour and first-void urine samples, although slightly higher Dpd levels were recorded in the first-void urine (19.6 ⫾ 5.6 nmol Dpd/mmol Cr vs. 20.5 ⫾ 5.8 nmol Dpd/mmol Cr, in respect, p ⬎ 0.05). In addition, a strong linear correlation in Dpd excretion was determined between 24-hour and first-void urine samples (r ⫽ 0.77, p ⬍ 0.05 ) (Figure 2). Discussion As an endogenous collagen marker, Dpd is closely associated with type I collagen which consists 90% of collagen of the organic bone matrix (3,5). It is excreted into urine in both free and peptide-bound forms (7). Measurement of peptide-bound forms requires hydrolyzation of the urine and is remarkably
TABLE 1 Demographic Characteristics of the Study Population (results expressed as mean ⫾ SD)
Age (months) Weight (kg) Height (cm) f-Dpd (first-void) (nmol/mmol Cr) f-Dpd (24-hour) (nmol/mmol Cr)
Overall (n ⫽ 62)
Boys (n ⫽ 35)
Girls (n ⫽ 27)
p
69.9 ⫾ 25.7 21.9 ⫾ 6.2 114.9 ⫾ 15.7 20.5 ⫾ 5.8
72.8 ⫾ 25.7 23.0 ⫾ 5.6 119.5 ⫾ 11.9 19.4 ⫾ 5.8
66.1 ⫾ 25.5 20.2 ⫾ 6.9 108 ⫾ 18.2 21.9 ⫾ 5.4
NSa NS p ⬍ 0.05 NS
19.6 ⫾ 5.6
18.6 ⫾ 5.3
20.9 ⫾ 5.8
NS
a
Not significant.
270
CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
URINARY DEOXYPYRIDINOLINE LEVELS
Figure 2 — Linear correlation graphic shows significant correlation between 24-hour and the first-void urine.
difficult. Whereas, by chemiluminescent enzyme immunoassay technique, f-Dpd would be measured easily without any pre-treatment of the urine specimen. In addition, some authors consider that direct immunoassay for urinary f-Dpd is as valuable as high performance liquid chromatography assay (10, 11). Gender-related changes in Dpd excretion were determined in several studies. The reported changes are frequently observed in the adolescence period, that seems to be caused by different timing of pubertal pattern in both genders (9,12–14). In the present study, there was no gender-related variations in Dpd excretion either in first-void urine or 24-hour samples. This might be a result of choosing the study population mainly from prepubertal period. In agreement with our consideration, similar gender-independent results were reported in prepubertal children (12,15). In our study, the primary goal was to evaluate the validity of f-Dpd excretion in single urine by comparing with 24-hour collection. Our results indicated that both collection methods have given well-correlated results, and f-Dpd values in the first-void urine are remarkable close to those in 24-hour. So far, Dpd assessment has been performed in several studies either in single urine (7,12,16 –18) or in 24-hour (3,19). There is no agreement on urinary sampling method between 24-hour or single sample, because majority of these studies are not comparable (7). In a few comparative studies, Abbiati et al. (20) compared free and total urinary Dpd levels in 24-hour and early morning urine samples in healthy 25 to 39-year-old volunteers. They found a significant difference in both total and free Dpd values between 24-hour and spot samples, as contrast to our results in the present study. Uebelhart and colleagues (21) noted significantly lower total Dpd value in single urine samples than 24-hour samples. Nevertheless, there is lack of comparative studies in childhood, since all of these studies were performed in adults. As a childhood study, Fujimoto et al. (9) investigated the relationship of total Dpd values in the first morning void urine and in 24-hour urine, and they CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
noted a strong correlation between the samples. Our results indicate that the correlation was also present in f-Dpd values. In agreement with us, Marowska et al. (15) suggested that single urine sampling provide maximum precision and sensitivity and Husain et al. (7) emphasized that there was no indication for 24-hour collection of urine sample unless quantification of bone resorption was required. Circadian pattern of Dpd excretion has been reported in several studies (15,22). Nevertheless, severity of these variations was controversial in these reports. Aoshima et al. (23) investigated urinary free, total, and peptide-bound Dpd excretion, and interestingly, they determined that f- Dpd has not been affected from circadian variations. In our study, slightly higher f-Dpd values were found in morning urine samples than those 24-hour collected. It seems to be caused by increased Dpd excretion during the night, however, the difference was not significant and may be negligible. Collagen cross-links have been considered as helpful in evaluation of some metabolic, endocrinologic, and neoplastic disorders (16,24,25). Deoxypyridinoline levels have been assessed in these studies either in serum or in 24-hour urine samples and both methods are invasive and have some difficulties such as catheterization or hospitalization, especially when performed in infants and young children. But, in our opinion, first-void f-Dpd values are wellcorrelated with those of 24-hour, and are genderindependent in the first decade of life. Therefore, it may safely be used in all first-decade children without requirement of invasive methods. Nevertheless, for detailed evaluation of urinary Dpd excretion in childhood, additional studies comparing all components of urinary Dpd in short-time versus long-time collected urine samples, plus in some pathological conditions are needed. References 1. Sharp CA, Oginni LM, Worsfold M, et al. Elevated collagen turnover in Nigerian children with calciumdeficiency rickets. Calcif Tissue Int 1997; 61: 87–94. 2. Zanze M, Souberbielle JC, Kindermans C, Rossignol C, Garabedian M. Procollagen propeptide and pyridinium cross-links as markers of type I collagen turnover: sex- and age-related changes in healthy children. J Clin Endocrinol Metab 1997; 82: 2971–77. 3. Rauch F, Schonau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994; 102: 94 –7. 4. Lund AM, Hansen M, Kollerup G, Jaul A, Teisner B, Skovby F. Collagen-derived markers of bone metabolism in osteogenesis imperfecta. Acta Paediatr 1998; 87: 1131–7. 5. Scott D, Abu Damir H, Buchan A, Robins SP. Factors affecting urinary pyridinoline and deoxypyridinoline excretion in the growing lamb. Bone 1993; 14: 807–11. 6. Endres DB, Rude RK. Mineral and Bone metabolism In: Burtis CA, Ashwood ER, Aldrich JE, Eds. Tietz fundamentals of clinical chemistry. Pp. 685–703 Philadelphia: W.B. Saunders Co, 1996. 271
SOYLU
7. Husain SM, Mughal Z, Williams G, et al. Urinary excretion of pyridinium crosslinks in healthy 4 –10 years olds. Arc Dis Child 1999; 80: 370 –3. 8. Seyedin SM, Kung VT, Daniloff YN, et al. Immunoassay for urinary pyridinoline: the new marker of bone resorption. J Bone Miner Res 1993; 8: 635– 41. 9. Fujimoto S, Kubo T, Tanaka H, Miura M, Seino Y. Urinary pyridinoline and deoxypyridinoline in healthy children and in children with growth hormone deficiency. J Clin Endocrinol Metab 1995; 80: 1922– 8. 10. Robins SP, Woitge H, Hesley R, Ju J, Seyedin S, Siebel MJ. Direct, enzyme-linked immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption. J Bone Miner Res 1994; 10: 1643–9. 11. Kent GN. Standardization of marker assays pyridinoline/deoxypyridinoline. Scand J Clin Lab Invest 1997; 57: 73–9. 12. Pereira RM, Falco V, Corrente JE, Chahade WH, Yoshinari NH. Abnormalities in the biochemical markers of bone turnover in children with juvenile chronic arthritis. Clin Exp Rheumatol 1999; 17: 251–5. 13. Mora S, Prinster C, Proverbio MC, et al. Urinary markers of bone turnover in healthy children and adolescents: age-related changes and effect of puberty. Calcif Tissue Int 1998; 63: 369 –74. 14. Conti A, Ferrero S, Giambona S, Sartorio A. Urinary free deoxypyridinoline levels during childhood. J Endocrinol Invest 1998; 21: 318 –22. 15. Marowska J, Kobylinska M, Lukaszkiewicz J, Talajko A, Rymkiewicz-Kluczynska B, Lorenc RS. Pyridinium crosslinks of collagen as a marker of bone resorption rates in children and adolescents: normal values and clinical application. Bone 1996; 19: 669 –77. 16. Ohishi T, Takahashi M, Kawana K, et al.Age-related changes of urinary pyridinoline and deoxypyridinoline in Japanese subjects. Clin Invest Med1993; 16: 319 – 25. 17. Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. Urinary excretion of pyridinoline crosslinks
272
ET AL.
18.
19.
20.
21.
22.
23.
24.
25.
correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis. J Bone Miner Res 1991; 6: 639 – 44. Tsukahara H, Watanabe Y, Hirano S, Tsubokura H, Kimura K, Mayumi M. Assessment of bone turnover in term and preterm newborns at birth: measurement of urinary collagen crosslink excretion. Early Hum Dev 1999; 53: 185–91. Abbiati G, Arrigoni M, Frignani S, Longoni A, Bartucci F, Castiglioni C. Effect of salmon calcitonin on deoxypyridinoline (Dpyr) urinary excretion in healthy volunteers. Calcif Tissue Int 1994; 55: 346 – 8. Abbiati G, Bartucci F, Longoni A, Fincato G, Galimberti S, Rigoldi M, Castiglioni C. Monitoring of free and total urinary pyridinoline in healthy volunteers: sample relationships between 24-h and fasting early morning urine concentrations. Bone Mineral 1993; 21: 9 –19. Uebelhart D, Schlemmer A, Johansen JS, Gineyts E, Christiansen C, Delmas PD. Effect of menopause and hormone replacement therapy on the urinary excretion of pyridinium cross-links. J Clin Endocrinol Metab 1991; 72: 367–73. Schlemmer A, Hassager C, Jensen SB, Christiansen C. Marked diurnal variation in urinary excretion of pyridinium cross-links in premenopausal women. J Clin Endocrinol Metab 1992; 74: 476 – 80. Aoshima H, Kushida K, Takahashi M, Ohishi T, Hoshino H, Suzuki M, Inoue T. Circadian variation of urinary type I collagen crosslinked C-telopeptide and free and peptide-bound forms of pyridinium crosslinks. Bone 1998; 22: 73– 8. Kipen Y, Will R, Strauss BJ, Morand EF. Urinary excretion of pyridinium cross-links of collagen in systemic lupus erythematosus. Clin Rheumatol 1998; 17: 271– 6. Nagasaka S, Sugimoto H, Nakamura T, et al. Antithyroid therapy improves bony manifestations and bone metabolic markers in patients with Graves’ thyrotoxicosis. Clin Endocrinol1997; 47: 215–21.
CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
PII S0009-9120(00)00072-2
Urinary Excretion of Deoxypyridinoline in 24-Hour and First-Void Samples in Healthy Turkish Children HANIFI SOYLU,1 S¸U¨KRU¨ ARAS,3 N. ONUR KUTLU1 , MU¨CAHIT EGˇRI,2 and KAMIL S¸AHIN4 Departments of 1Pediatrics and 2 Public Health, I˙no¨nu¨ University, Turgut O¨zal Medical Center, Malatya, Turkey; 3Department of Biochemistry, Vakıf Gureba Teaching Hospital, Istanbul, Turkey; 4 Department of Pediatrics, Anadolu Hospital, Istanbul, Turkey Objectives: Collagen cross-links are formed during the maturation process of bone matrix. They have been considered as valuable markers in some metabolic, endocrinologic, and neoplastic bone disorders. As an advantage, it can be measured in urine as well as in serum samples. However, the excretion characteristics remains controversial. Design and methods: We investigated urinary free deoxypyridinoline (f-Dpd) excretion in first-void urine samples and in 24-hour collections in healthy Turkish children. We also evaluated the possible correlations and gender-related differences in Dpd excretion between these sampling methods. Both urine samples of 62 subjects (aged from 31 to 120 months) were analyzed by Immulite chemiluminescent technique. Results: There were no remarkable differences in f-Dpd excretion between first-void and 24-hour urine samples, although f-Dpd values of the first-void samples were slightly higher (Dpd: creatinine, mean ⫾ SD, 20.5 ⫾ 5.8 nmol/mmol vs. 19.6 ⫾ 5.6 nmol/mmol, respectively, p ⬎ 0.05). A strong linear correlation was found between 24-hour and first-void urine f-Dpd excretions (r ⫽ 0.77, p ⬍ 0.05). In addition, f-Dpd showed no gender-related differences between boys and girls in either 24-hour or first-void urine samples (p ⬎ 0.05). Conclusions: Because of difficulties in long-time urine collection in infants and young children, f-Dpd assessment in first-void single urine samples is an easy, safe, and non-invasive method. Copyright © 2000 The Canadian Society of Clinical Chemists
KEY WORDS: deoxypyridinoline; childhood; first-void single urine.
(Dpd) occur and stabilize mature collagen chain within the extracellular matrix (2). The cross-links are formed during the maturation, not the biosynthesis, of collagen and release into circulation and excreted by urine without metabolization (3– 6). This is a continuous process in normal growth and bone remodeling (3– 6). Some markers for bone metabolism such as osteocalcin, bone specific alkaline phosphatase, and propeptides that derived from type I collagen, have disadvantages such as requirement of serum assessment, whereas Pyd and Dpd can be measured in either serum or urine (7). Some authors had preferred single urine assessment in their studies, however, the others collected 24-hour one (7,8). The single urine assessment is easier and more practical. Twenty-four hour urine collection is troublesome and often requires hospitalization especially in infants and young children. However, there is only a few comparative data between these methods in childhood (7,9). To clarify this situation, we aimed to compare free Dpd (f-Dpd) levels between urine samples of first-void and 24-hour in order to determine whether the first-void urine has any value in determination of f-Dpd excretion.
Introduction he organic matrix of bone is a complex-organized structure that undergoes mineralization. It is largely the product of the osteoblasts, and consisted mainly of type I collagen, which has functional cross-linked propeptides that are located to the telopeptide region (1). Their lysyl or hydroxylysyl components are converted by lysyl hydroxylase or lysyl oxidase to aldehydes during the maturation. Thus, pyridinoline (Pyd) and deoxypyridinoline
T
Correspondence: Dr. Hanifi Soylu, I˙no¨nu¨ University, ¨ zal Medical Center, Department of Pediatrics, Turgut O Malatya, Turkey. E-mail: [email protected]. Manuscript received December 22, 1999; revised February 29, 2000; accepted March 23, 2000. CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
Material and methods SUBJECTS Sixty-two healthy Turkish children were enrolled in this prospective study. By history, clinical examination, and routine laboratory assessment, none of the subjects had any evidence of bone, hepatic, renal, or endocrine diseases or malnutrition, and none of them was taking medication that acts on bone metabolism. The parents were informed about the aim and method of the study. Age (months), height (cm), and weight (kg) of each child were recorded. No diet or exercise restriction was done prior to the study. 269
SOYLU
URINE
ET AL.
COLLECTION AND ANALYTICAL METHOD
Two different samples were obtained, one from a 24-hour sample and the other from the first morning void urine sample. The parents were educated about how the urine collection should be performed. The urine bags were used for collection of the samples when needed in young children. The urine sample was centrifuged, and the supernatant was stored without preservative at ⫺20 °C until the analysis. Deoxypyridinoline measurement was performed by chemiluminescent enzyme immunoassay (CEI) according to Immulite method (Immulite Pyrilinks-D, Catalog No:LKPD1 Diagnostic Products Corporation, CA, USA). This test is a solid phase containing a polystyrene bead that is coated with monoclonal antibody specific for Dpd. At the study phase, the urine specimen and alkaline phosphatase-conjugated Dpd were simultaneously introduced into the test unit and incubated for 30 min at 37 °C with intermittent agitation for competition of the sample with the enzymelabeled Dpd. Unbound enzyme conjugate was then removed by a centrifugal wash with distilled water, afterwards concentration was measured by the luminometer. Test results were normalized to the urinary creatinine concentration to correct for variations in urinary flow. Urinary creatinine was assessed by BM CREA creatinine kit (Boehringer-Mannheim, Germany, Hi Co Creatinine, Catalog No: 1040847) in Hitachi 717 biochemical analytical system (Boehringer-Mannheim Hitachi, Germany-Japan). Each urine sample was diluted as a ratio of 1/10. Each Dpd result (nmol/L) was divided by the sample’s creatinine (Cr) value (mmol/L), therefore, the results were expressed as nmol Dpd/mmol Cr. STATISTICAL
METHODS
The SPSS computerized statistical software (SPSS, version 7.5.1, Statistical Package for the Social Sciences Inc., Chicago, IL, USA) was used for the statistical analyses. Differences between mean Dpd values of both urinary samples and differences between both sexes were determined by Student’s t test. The correlation between 24-hour and first-void urine samples was assessed by Pearson correlation test. Statistical significance was ascribed when p was equal to or less than 0.05.
Figure 1 — Urinary f-Dpd excretion in 24-hour and the first-void urine samples in girls and boys (p ⬎ 0.05).
Results The study population consisted of 62 children (35 boys, 27 girls) aged 31 to 120 months (mean age ⫾ SD; 69.88 ⫾ 25.65 months). There were no statistically significant differences between ages and weights of both sexes, except heights. The heights of boys were significantly taller than girls (119.5 ⫾ 11.9 cm vs. 108 ⫾ 18.2 cm, in respect, p ⬍ 0.05). Urinary Dpd levels were similar between both genders in the first-void urine (boys; 19.4 ⫾ 5.8 nmol Dpd/mmol Cr vs. girls; 21.9 ⫾ 5.4 nmol Dpd/mmol Cr, p ⬎ 0.05) and in the 24-hour urine samples (boys; 18.6 ⫾ 5.3 nmol Dpd/mmol Cr vs. girls; 20.9 ⫾ 5.4 nmol Dpd/mmol Cr, p ⬎ 0.05) (Figure 1). The physical characteristics of study population and statistical features were shown on Table 1. We did not observe any significant difference in Dpd values between 24-hour and first-void urine samples, although slightly higher Dpd levels were recorded in the first-void urine (19.6 ⫾ 5.6 nmol Dpd/mmol Cr vs. 20.5 ⫾ 5.8 nmol Dpd/mmol Cr, in respect, p ⬎ 0.05). In addition, a strong linear correlation in Dpd excretion was determined between 24-hour and first-void urine samples (r ⫽ 0.77, p ⬍ 0.05 ) (Figure 2). Discussion As an endogenous collagen marker, Dpd is closely associated with type I collagen which consists 90% of collagen of the organic bone matrix (3,5). It is excreted into urine in both free and peptide-bound forms (7). Measurement of peptide-bound forms requires hydrolyzation of the urine and is remarkably
TABLE 1 Demographic Characteristics of the Study Population (results expressed as mean ⫾ SD)
Age (months) Weight (kg) Height (cm) f-Dpd (first-void) (nmol/mmol Cr) f-Dpd (24-hour) (nmol/mmol Cr)
Overall (n ⫽ 62)
Boys (n ⫽ 35)
Girls (n ⫽ 27)
p
69.9 ⫾ 25.7 21.9 ⫾ 6.2 114.9 ⫾ 15.7 20.5 ⫾ 5.8
72.8 ⫾ 25.7 23.0 ⫾ 5.6 119.5 ⫾ 11.9 19.4 ⫾ 5.8
66.1 ⫾ 25.5 20.2 ⫾ 6.9 108 ⫾ 18.2 21.9 ⫾ 5.4
NSa NS p ⬍ 0.05 NS
19.6 ⫾ 5.6
18.6 ⫾ 5.3
20.9 ⫾ 5.8
NS
a
Not significant.
270
CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
URINARY DEOXYPYRIDINOLINE LEVELS
Figure 2 — Linear correlation graphic shows significant correlation between 24-hour and the first-void urine.
difficult. Whereas, by chemiluminescent enzyme immunoassay technique, f-Dpd would be measured easily without any pre-treatment of the urine specimen. In addition, some authors consider that direct immunoassay for urinary f-Dpd is as valuable as high performance liquid chromatography assay (10, 11). Gender-related changes in Dpd excretion were determined in several studies. The reported changes are frequently observed in the adolescence period, that seems to be caused by different timing of pubertal pattern in both genders (9,12–14). In the present study, there was no gender-related variations in Dpd excretion either in first-void urine or 24-hour samples. This might be a result of choosing the study population mainly from prepubertal period. In agreement with our consideration, similar gender-independent results were reported in prepubertal children (12,15). In our study, the primary goal was to evaluate the validity of f-Dpd excretion in single urine by comparing with 24-hour collection. Our results indicated that both collection methods have given well-correlated results, and f-Dpd values in the first-void urine are remarkable close to those in 24-hour. So far, Dpd assessment has been performed in several studies either in single urine (7,12,16 –18) or in 24-hour (3,19). There is no agreement on urinary sampling method between 24-hour or single sample, because majority of these studies are not comparable (7). In a few comparative studies, Abbiati et al. (20) compared free and total urinary Dpd levels in 24-hour and early morning urine samples in healthy 25 to 39-year-old volunteers. They found a significant difference in both total and free Dpd values between 24-hour and spot samples, as contrast to our results in the present study. Uebelhart and colleagues (21) noted significantly lower total Dpd value in single urine samples than 24-hour samples. Nevertheless, there is lack of comparative studies in childhood, since all of these studies were performed in adults. As a childhood study, Fujimoto et al. (9) investigated the relationship of total Dpd values in the first morning void urine and in 24-hour urine, and they CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000
noted a strong correlation between the samples. Our results indicate that the correlation was also present in f-Dpd values. In agreement with us, Marowska et al. (15) suggested that single urine sampling provide maximum precision and sensitivity and Husain et al. (7) emphasized that there was no indication for 24-hour collection of urine sample unless quantification of bone resorption was required. Circadian pattern of Dpd excretion has been reported in several studies (15,22). Nevertheless, severity of these variations was controversial in these reports. Aoshima et al. (23) investigated urinary free, total, and peptide-bound Dpd excretion, and interestingly, they determined that f- Dpd has not been affected from circadian variations. In our study, slightly higher f-Dpd values were found in morning urine samples than those 24-hour collected. It seems to be caused by increased Dpd excretion during the night, however, the difference was not significant and may be negligible. Collagen cross-links have been considered as helpful in evaluation of some metabolic, endocrinologic, and neoplastic disorders (16,24,25). Deoxypyridinoline levels have been assessed in these studies either in serum or in 24-hour urine samples and both methods are invasive and have some difficulties such as catheterization or hospitalization, especially when performed in infants and young children. But, in our opinion, first-void f-Dpd values are wellcorrelated with those of 24-hour, and are genderindependent in the first decade of life. Therefore, it may safely be used in all first-decade children without requirement of invasive methods. Nevertheless, for detailed evaluation of urinary Dpd excretion in childhood, additional studies comparing all components of urinary Dpd in short-time versus long-time collected urine samples, plus in some pathological conditions are needed. References 1. Sharp CA, Oginni LM, Worsfold M, et al. Elevated collagen turnover in Nigerian children with calciumdeficiency rickets. Calcif Tissue Int 1997; 61: 87–94. 2. Zanze M, Souberbielle JC, Kindermans C, Rossignol C, Garabedian M. Procollagen propeptide and pyridinium cross-links as markers of type I collagen turnover: sex- and age-related changes in healthy children. J Clin Endocrinol Metab 1997; 82: 2971–77. 3. Rauch F, Schonau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994; 102: 94 –7. 4. Lund AM, Hansen M, Kollerup G, Jaul A, Teisner B, Skovby F. Collagen-derived markers of bone metabolism in osteogenesis imperfecta. Acta Paediatr 1998; 87: 1131–7. 5. Scott D, Abu Damir H, Buchan A, Robins SP. Factors affecting urinary pyridinoline and deoxypyridinoline excretion in the growing lamb. Bone 1993; 14: 807–11. 6. Endres DB, Rude RK. Mineral and Bone metabolism In: Burtis CA, Ashwood ER, Aldrich JE, Eds. Tietz fundamentals of clinical chemistry. Pp. 685–703 Philadelphia: W.B. Saunders Co, 1996. 271
SOYLU
7. Husain SM, Mughal Z, Williams G, et al. Urinary excretion of pyridinium crosslinks in healthy 4 –10 years olds. Arc Dis Child 1999; 80: 370 –3. 8. Seyedin SM, Kung VT, Daniloff YN, et al. Immunoassay for urinary pyridinoline: the new marker of bone resorption. J Bone Miner Res 1993; 8: 635– 41. 9. Fujimoto S, Kubo T, Tanaka H, Miura M, Seino Y. Urinary pyridinoline and deoxypyridinoline in healthy children and in children with growth hormone deficiency. J Clin Endocrinol Metab 1995; 80: 1922– 8. 10. Robins SP, Woitge H, Hesley R, Ju J, Seyedin S, Siebel MJ. Direct, enzyme-linked immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption. J Bone Miner Res 1994; 10: 1643–9. 11. Kent GN. Standardization of marker assays pyridinoline/deoxypyridinoline. Scand J Clin Lab Invest 1997; 57: 73–9. 12. Pereira RM, Falco V, Corrente JE, Chahade WH, Yoshinari NH. Abnormalities in the biochemical markers of bone turnover in children with juvenile chronic arthritis. Clin Exp Rheumatol 1999; 17: 251–5. 13. Mora S, Prinster C, Proverbio MC, et al. Urinary markers of bone turnover in healthy children and adolescents: age-related changes and effect of puberty. Calcif Tissue Int 1998; 63: 369 –74. 14. Conti A, Ferrero S, Giambona S, Sartorio A. Urinary free deoxypyridinoline levels during childhood. J Endocrinol Invest 1998; 21: 318 –22. 15. Marowska J, Kobylinska M, Lukaszkiewicz J, Talajko A, Rymkiewicz-Kluczynska B, Lorenc RS. Pyridinium crosslinks of collagen as a marker of bone resorption rates in children and adolescents: normal values and clinical application. Bone 1996; 19: 669 –77. 16. Ohishi T, Takahashi M, Kawana K, et al.Age-related changes of urinary pyridinoline and deoxypyridinoline in Japanese subjects. Clin Invest Med1993; 16: 319 – 25. 17. Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. Urinary excretion of pyridinoline crosslinks
272
ET AL.
18.
19.
20.
21.
22.
23.
24.
25.
correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis. J Bone Miner Res 1991; 6: 639 – 44. Tsukahara H, Watanabe Y, Hirano S, Tsubokura H, Kimura K, Mayumi M. Assessment of bone turnover in term and preterm newborns at birth: measurement of urinary collagen crosslink excretion. Early Hum Dev 1999; 53: 185–91. Abbiati G, Arrigoni M, Frignani S, Longoni A, Bartucci F, Castiglioni C. Effect of salmon calcitonin on deoxypyridinoline (Dpyr) urinary excretion in healthy volunteers. Calcif Tissue Int 1994; 55: 346 – 8. Abbiati G, Bartucci F, Longoni A, Fincato G, Galimberti S, Rigoldi M, Castiglioni C. Monitoring of free and total urinary pyridinoline in healthy volunteers: sample relationships between 24-h and fasting early morning urine concentrations. Bone Mineral 1993; 21: 9 –19. Uebelhart D, Schlemmer A, Johansen JS, Gineyts E, Christiansen C, Delmas PD. Effect of menopause and hormone replacement therapy on the urinary excretion of pyridinium cross-links. J Clin Endocrinol Metab 1991; 72: 367–73. Schlemmer A, Hassager C, Jensen SB, Christiansen C. Marked diurnal variation in urinary excretion of pyridinium cross-links in premenopausal women. J Clin Endocrinol Metab 1992; 74: 476 – 80. Aoshima H, Kushida K, Takahashi M, Ohishi T, Hoshino H, Suzuki M, Inoue T. Circadian variation of urinary type I collagen crosslinked C-telopeptide and free and peptide-bound forms of pyridinium crosslinks. Bone 1998; 22: 73– 8. Kipen Y, Will R, Strauss BJ, Morand EF. Urinary excretion of pyridinium cross-links of collagen in systemic lupus erythematosus. Clin Rheumatol 1998; 17: 271– 6. Nagasaka S, Sugimoto H, Nakamura T, et al. Antithyroid therapy improves bony manifestations and bone metabolic markers in patients with Graves’ thyrotoxicosis. Clin Endocrinol1997; 47: 215–21.
CLINICAL BIOCHEMISTRY, VOLUME 33, JUNE 2000