or quality in early postmenopausal women

RESEARCH LETTERS

Association Between Serum Undercarboxylated Osteocalcin and Bone Density and/or Quality in Early Postmenopausal Women

Blood for measurement of ucOC, OC, and carboxy-terminal telopeptide of type I procollagen (ICTP) was collected in the morning, after overnight fasting. Samples were immediately centrifuged and stored at ⫺80°C until analyzed. A fasting spot of the 2-h second morning urine was collected from all women for the measurement of free deoxypyridinoline (DPD) concentrations. The values of urinary DPD were expressed as nanomoles per millimole of urinary creatinine.

INTRODUCTION Analytical Methods Vitamin K functions as a cofactor for carboxylase, a microsomal enzyme that facilitates the posttranslational conversion of glutamyl to ␥-carboxy glutamyl residues of proteins, including osteocalcin. Circulating undercarboxylated osteocalcin (ucOC) is thus a valuable nutrition marker reflecting skeletal provision with vitamins K and D.1,2 Some studies have demonstrated the strong relation of this parameter to the risk of fractures or to hip bone mineral density (BMD) in elderly women.2–7 The aim of the present study was to analyze the association between serum ucOC and BMD, calcaneal ultrasound parameters, and fracture rate in early postmenopausal women. To increase the sensitivity of the analysis, serum ucOC was corrected for total osteocalcin (OC) and expressed as the ratio of ucOC to OC.

MATERIALS AND METHODS This was a case control study performed in 113 postmenopausal women from the general Czech population who were screened for diseases associated with menopause. The mean age (⫾ standard deviation) of these women was 62.4 (⫾ 9.3) y, mean years since menopause was 13.0 (⫾ 7.8), and mean BMI was 25.7 (⫾ 3.6) kg/m2. Of these, 33 were healthy (age, 60.1 ⫾ 10.3 y), 65 were osteoporotic (T score ⬍⫺2.5, i.e., more than 2.5 standard deviations below the peak bone mass value in the young adult population; age, 63.6 ⫾ 7.8 y), and 16 women were osteopenic (T score ⫺2.0 to ⫺2.5). Twenty-nine women had a history of one or more fractures. Fifteen fractures were localized at the distal forearm, seven at the tibia, six at the humerus, and three at the ribs. From two femoral fractures, only one was localized at the hip. The time since the last fracture exceeded 2 y in all probands. The study group did not include any alcoholics, heavy smokers (more than seven cigarettes a day), and conditions such as psychosis, endocrinopathy, malignancy, or other serious disease leading to pathologic fracture. None of the women had early or late menarche or premature menopause (before age 45 y). Women with unclear menopausal status were not included in the study. The prior menstrual history of healthy, osteoporotic, and osteopenic subjects had been regular (11 to 13 cycles/y). None had been treated with vitamins K and D or with calciotropic drugs before the study. No woman was markedly underweight or obese, and all had unrestricted calcium, caloric, protein, and green vegetable intakes. The daily life of the probands was usual in physical activity. Informed consent was obtained from all subjects, and all procedures were approved by the Ethical Committee of the Institute of Endocrinology, Prague.

This study was funded by grant NB7391-3 and from the Internal Grant Agency of the Ministry of Health of the Czech Republic. Correspondence to: Ivana Zˇofkova´, MD, PhD, DSc, Institute of Endocrinology, Prague, Czech Republic. E-mail: [email protected] Nutrition 19:1001–1005, 2003 ©Elsevier Inc., 2003. Printed in the United States. All rights reserved.

Serum ucOC concentration was assessed with a monoclonal antibody (enzyme-linked immunosorbent assay [ELISA] kit, Takara Shuzo Company, Ltd., Tokyo, Japan) without binding to hydroxyapatite. Serum total OC was determined by electrochemiluminescence immunoassay with the Elecsis-2010 analyzer (Roche, Switzerland). This method recognizes fully carboxylated N-terminal fragments in addition to the intact molecule. Serum ucOC was interpreted in terms of the total OC with the ratio of ucOC to OC. ICTP was measured with an radioimmunoassay kit from Orion Diagnostica (Espoo, Finland). Free DPD in the urine was determined by ELISA (Metra Biosystems, Palo Alto, CA, USA). Urinary creatinine was estimated photometrically with an semiautomatic analyzer (Merck VitaLab-Eclipse, Germay). Duplicate measurements were used to form mean values. The interassay coefficients of variation were as follows: 8.3% for ucOC, 14.0% for total OC, 7.4% for ICTP, 4.0% for DPD, and 6.0% for creatinine. BMD at the hip and at the lumbar spine (g/cm2) was estimated by dual energy x-ray absorptiometry (Hologic, QDR-2000, Waltham, MA, USA). Bone quality was measured by calcaneal broadband ultrasound attenuation (BUA; dB/MHz), speed of sound (SOS; m/s), stiffness index (0.67 ⫻ BUA ⫹ 0.28 ⫻ SOS ⫺ 420; measured with an Achilles ultrasound device), and fracture rate. Sensitivity and specificity of these methods were as follows: 0.75% and 0.64% for BMD, 0.75% and 0.73% for BUA, and 0.75% and 0.64% for SOS, respectively. The method precisions were 2% for BMD, 4% for BUA, and 0.5% for SOS. Statistical Evaluation Relations between variables (including years since menopause) were investigated with factor analysis (FA). The FA was used because of its capability to show the inherent structure of the relations between the variables. In contrast to multiple regression, it is truly multidimensional. It evaluates the relation between independent (orthogonal) so-called variates or factors that are computed to obtain the maximum correlation between them and the corresponding variables. The factors obtained (mostly after rotation) could be commonly interpreted from the biological viewpoint, and they are frequently more informative than the original variables. Moreover, from the blur of many more or less correlated variables, one can obtain a considerably smaller number of easily interpreted “variates” (factors), thereby enabling evaluation of the contribution of each original variable to the property expressed by the factor. In general, one variable could contribute to more than one factor and the measure of the contribution of the variable could be estimated with such an approach. Sometimes the variable can be highly correlated with the factor (factor loading close to 1), and thus the other, less correlating variables need not be involved to explain the property expressed by the factor. From the matrix of factor loadings representing the correlation coefficients between 0899-9007/03/$30.00

1002

Research Letters

Nutrition Volume 19, Numbers 11/12, 2003 TABLE I.

PHYSICAL, BIOCHEMICAL, AND ANTHROPOMETRIC CHARACTERISTICS OF INVESTIGATED WOMEN

Variable (normal range) Density, structure, and fragility Number of fractures BMI (kg/m2) BMD at the hip (g/cm2) BMD at the spine (g/cm2) BUA (db/MHz) Elasticity Stiffness index SOS (m/s) Serum (ucOC/OC) Remodeling Serum ICTP (1.8–5.0 ng/mL) Urinary DPD (2 h) (3.0–7.4 nM/mM) Serum ucOC (1.7–9.9 ng/mL) Serum OC (11.0–43.0 ng/mL) Years since menopause

Count

Mean

SD

Median

Lower quartile

Upper quartile

Minimum

Maximum

111 114 112 112 114

0.495 25.8 0.803 0.857 68.4

0.971 3.7 0.129 0.170 18.8

0.000 25.5 0.776 0.850 65.0

0.000 23.2 0.720 0.755 54.0

1.000 27.9 0.883 0.941 80.0

0.000 19.0 0.500 0.000 36.0

4.000 37.0 1.160 1.320 134.0

89 89 87

74.7 1603 0.122

22.4 50 0.105

72.5 1601 0.096

57.7 1566 0.072

85.3 1636 0.152

29.7 1503 0.008

153.4 1768 0.820

89 86 88 88 114

3.81 8.14 2.77 21.6 15.3

1.54 6.59 3.04 11.9 9.0

3.48 6.95 1.88 18.2 14.0

2.83 5.80 1.02 13.5 8.0

4.40 8.90 3.22 26.5 21.0

1.52 3.20 0.14 5.6 0.5

10.40 64.80 21.40 75.7 40.0

BMD, bone mineral density; BMI, body mass index; BUA, broadband ultrasound attenuation; DPD, deoxypyridinoline; ICTP, carboxy-terminal telopeptide of type I procollagen; OC, osteocalcin; SD, standard deviation; SOS, speed of sound; ucOC, undercarboxylated osteocalcin

the factors and the individual variables, the possible regression models usually could be estimated but not vice versa. Only in the special case, when all variables do not share variability with more than one factor, the model of the FA can be replaced by a set of multiple regression models. However, even in this case, it could be a problem to determine which variable is dependent and which is independent. Instead of laborious and frequently aimless searching for the best set of regression models, the entire structure of the relation can be resolved with a single analysis. The additional advantage of FA is the fact that the variables are usually standardized before analysis and, thus being independent of the scale, can be more easily interpreted.

RESULTS Anthropometric, physical, and biochemical characteristics of the subjects are shown in Table I. FA including years since menopause and body mass index in the model (Table II) associated the ucOC/OC ratio to the factor of elasticity (factor loading, 0.328), although this relation was not very strong, explaining only about 5% variability of the factor. The ucOC/OC ratio did not cohere with the factors of bone density, structure, or fragility (dual energy x-ray absorptiometry, BUA, stiffness, and n fractures). No association was observed between the ucOC/OC ratio and characteristics of bone remodeling (OC, DPD, and ICTP).

DISCUSSION In the present study, the serum ucOC/OC ratio was associated with the factor of elasticity, but not with other parameters of bone quality or BMD. These results are similar to the data of Liu et al.3 who found a relation between serum ucOC and SOS. Thus, the present study, in agreement with the results of Liu et al., supports the hypothesis that serum ucOC is related more to some parameters of bone quality than to BMD. No associations were found between serum ucOC/OC ratio and fracture rate, which contrasts with the data obtained by others. The extensive prospective cohort study showed that ucOC predicts hip

fracture risk independently of femoral neck BMD in women older than 75 y.2 A prospective study found that elderly women with an increased serum ucOC have a six-fold increased risk of sustaining a hip fracture during follow-up as compared with those with normal serum ucOC.6 Moreover, logistic regression has indicated the correlation of serum ucOC to hip fractures when age and parathyroid hormone levels are included in the model.7 The predictive importance of serum ucOC and/or ucOC/OC ratio for the occurrence of fractures in elderly subjects has been suggested by others.8 The absence of a relation between serum ucOC/OC and BMD in our study contrasts with the results obtained in a comparable sample of elderly women.5 The latter study showed that ucOC has the highest predictive value for BMD when age and body weight are included in the equation, indicating that serum ucOC is a determinant of BMD of the hip in elderly women. These studies differ from the present investigation in some important aspects. First, the study groups in the cited publications comprised women older than 70 y with hip fractures. In contrast, our postmenopausal women were substantially younger, and only one had a history of hip fracture, whereas the others had a spectrum of fractures of different localizations. Thus serum ucOC appears to be associated with particular types of fractures. Further, the method used for measurement of serum ucOC should be taken into account. In most of the cited studies, serum ucOC was determined by hydroxyapatite binding assay, whereas in the present study ELISA was used. Cross-reactivity of ELISA for total OC has been found to be approximately 5%.2 Despite that finding, ELISA discriminates between ucOC and total OC very well.9 A more important limiting factor is increased reactivity of antibody specific for ucOC to large N-terminal fragments, which may complicate interpretation of the results obtained. Our study showed no association between total OC and BMD, even though the relation of this parameter to bone remodeling is evident. This is in contrast with the data published by Ravn et al.10 showing an association between serum N-terminal mid fragment of OC and bone density at the lumbar spine, proximal femur, and in the distal forearm in pre- and postmenopausal women. Conversely, negative data were obtained in EPIDOS study.2 The

Nutrition Volume 19, Numbers 11/12, 2003

Research Letters

1003

TABLE II. FACTOR ANALYSIS OF THE RELATIONS AMONG INDICES OF BONE DENSITY AND/OR QUALITY, ANTHROPOMETRIC CHARACTERISTICS, AND PARAMETERS OF BONE REMODELING CALCULATED FROM SPERMAN’S CORRELATION MATRIX* Factor loadings Factor 1

Factor 2

Factor 3

Variance Factor no.

Eigenvalue

%

Cumulative %

Variable

Communality

Density, structure, and fragility

Elasticity

Remodeling

1 2 3 4 5 6 7 8 9 10 11 12

3.33 1.58 1.21 0.42 0.38 0.15 0.05 0.00 0.00 0.00 0.00 0.00

46.8 22.2 17.0 5.9 5.3 2.1 0.7 0.0 0.0 0.0 0.0 0.0

46.8 69.0 86.0 91.9 97.2 99.3 100.0 100.0 100.0 100.0 100.0 100.0

Number of fractures BMI BMD at the hip BMD at the spine BUA Stiffness index SOS Serum (ucOC/OC) Serum ICTP Urinary DPD (2 h) Serum OC Years since menopause

0.127 0.248 0.651 0.428 0.800 0.970 0.867 0.108 0.488 0.321 0.501 0.601

⫺0.327 0.474 0.793 0.623 0.745 0.448 0.010 0.015 0.154 0.008 ⫺0.045 ⫺0.483

⫺0.110 ⫺0.126 0.144 0.044 0.474 0.873 0.931 0.328 0.106 ⫺0.100 0.077 ⫺0.281

0.090 0.085 ⫺0.023 0.195 ⫺0.143 ⫺0.089 0.018 0.020 0.673 0.558 0.702 0.537

* VARIMAX rotation followed factor analysis. Factor loadings represent correlation coefficients between factors and variables, and communality is a measure of the shared variability between the variable and all remaining variables. The number of factors was determined with Kaiser’s criterion (only the factors with eigenvalue greater than 1 were included). In this case it means that 86% of the total variability from 12 original variables was explained by only three factors. The boldface text denotes the relevant information in individual statistical parameters. BMD, bone mineral density; BUA, broadband ultrasound attenuation; DPD, deoxypyridinoline; ICTP, carboxy-terminal telopeptide of type 1 procollagen; OC, osteocalcin; SOS, speed of sound; ucOC, undercarboxylated osteocalcin

problem of cross-reactivity between OC and the molecule fragments also may have limited the interpretation of this parameter. To summarize, the present study did not confirm associations between serum ucOC and bone density in early postmenopausal women. However, it indicated a moderate relation of ucOC to bone elasticity. Association analyses in women subdivided according to fracture type are needed.

Ivana Zˇofkova´, MD, PhD, DSc Martin Hill, PhD, DSc Vladimı´r Palicˇka, MD, PhD Institute of Endocrinology Prague, Czech Republic Institute of Clinical Biochemistry and Diagnostics Charles University Teaching Hospital Hradec, Kra´love´, Czech Republic REFERENCES 1. Sokoll LJ, Booth SL, O’Brien ME, Davidson KW, Tsaioun KI, Sadowski JA. Changes in serum osteocalcin, plasma phylloquinone, and urinary ␥-carboxy glutamic acid in response to altered intakes of dietary phylloquinone in human subjects. Am J Clin Nutr 1981;65:779 2. Vergnaud P, Garnero P, Meunier PJ, Bre´ art G, Kamihagi K, Delmas PD. Undercarboxylated osteocalcin measured with a specific immunoassay predicts hip fracture in elderly women: the EPIDOS study. J Clin Endocrinol Metab 1997;82:719 3. Liu G, Peacock M. Age-related changes in serum undercarboxylated osteocalcin and its relationships with bone density, bone quality, and hip fracture. Calcif Tissue Int 1998;62:286 4. Ferland G. The vitamin K– dependent proteins: an update. Nutr Rev 1998;56:223 5. Szulc P, Arlot M, Chapuy M-C, Duboeuf F, Meunier PJ, Delmas PD. Serum undercarboxylated osteocalcin correlates with hip bone mineral density in elderly women. J Bone Miner Res 1994;9:1591

6. Szulc P, Chapuy M-C, Meunier PJ, Delmas PD. Serum undercarboxylated osteocalcin is a marker of the risk of hip fracture in elderly women. J Clin Invest 1993;91:1769 7. Szulc P, Chapuy M-C, Meunier PJ, Delmas PD. Serum undercarboxylated osteocalcin is a marker of the risk of hip fracture: a three year follow-up study. Bone 1996;18:487 8. Luukinen H, Ka¨ ko¨ nen S-M, Pettersson K, et al. Strong prediction of fractures among older adults by the ratio of carboxylated to total serum osteocalcin. J Bone Miner Res 2000;15:2473 9. Grundberg CM, Nieman SD, Abrams S, Rosen H. Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin. J Clin Endocrinol Metab 1998;83:3258 10. Ravn P, Fledelius C, Rosenquist C, Overgaard K, Christiansen C. High bone turnover is associated with low bone mass in both pre- and postmenopausal women. Bone 1996;19:291

Zinc, Copper, and Iron in Plasma and Tissues After Intestinal Ischemia and Reperfusion in the Rat INTRODUCTION The plasma and tissue content of trace minerals such as zinc, copper, and iron are significantly affected by stress,1 burns,2 and

The research reported in this article was supported by the Human Nutrition Research Funds of Texas. Correspondence to: V. Bruce Grossie, Jr., PhD, Department of Nutrition and Food Sciences, Texas Woman’s University, PO Box 425888, Denton, TX 76204-5888, USA. E-mail: [email protected]