RETRACTED: Menatetrenone and vitamin D2 with calcium supplements prevent nonvertebral fracture in elderly women with Alzheimer's disease

Bone 36 (2005) 61 – 68 www.elsevier.com/locate/bone

Menatetrenone and vitamin D2 with calcium supplements prevent nonvertebral fracture in elderly women with Alzheimer’s disease Yoshihiro Satoa,b,*, Tomohiro Kanokoc, Kei Satohd, Jun Iwamotoe a Department of Neurology, Mitate Hospital, Tagawa 826-0041, Japan Department of Neurology, Futase Social Insurance Hospital, Iizuka 820-0054, Japan c Department of Rehabilitation Medicine, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan d Department of Vascular Biology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan e Department of Sport Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan b

Received 21 June 2004; revised 23 September 2004; accepted 29 September 2004 Available online 24 November 2004

Abstract A high incidence of fractures, particularly of the hip, represents an important problem in patients with Alzheimer’s disease (AD), who are prone to falls and may have osteoporosis. We previously showed deficiency of vitamins D and K1 causes reduced bone mineral density (BMD) in female AD patients. The present study was undertaken to address the possibility that treatment with vitamin K2 (menatetrenone; MK-4) may maintain BMD and reduce the incidence of nonvertebral fractures in elderly female patients with AD. In a random and prospective study of AD patients, 100 patients received 45 mg menatetrenone, 1000 IU ergocalciferol and 600 mg calcium daily for 2 years, and the remaining 100 (untreated group) did not. At baseline, patients of both groups showed vitamin D and K1 deficiencies. They also had high serum levels of parathyroid hormone (PTH) and Glu osteocalcin (OC) and low serum ionized calcium, indicating that vitamin D deficiency stimulates compensatory PTH secretion. During the 2-year study period, BMD in the second metacarpals increased by 2.3% in the treated group and decreased by 5.2% in the untreated group (P b 0.0001). Serum levels of vitamin K2 and 25-hydroxyvitamin D increased by 284.9% and 147.9%, respectively, in the treated group. Correspondingly, a significant decrease in Glu OC and PTH were observed, in association with an increased calcium levels, in the treated group. Twenty-two patients in the untreated group sustained nonvertebral fractures (15 with hip fractures, two fractures each at the distal forearm and the proximal femur, each one fracture at the proximal humerus, ribs, and pelvis), and three fractures (2 with hip fractures, one fracture at the proximal femur) occurred among the treated patients (P = 0.0003; odds ratio = 7.5). Treatment with MK-4 and vitamin D2 with calcium supplements increases the BMD in elderly female patients with AD and leads to the prevention of nonvertebral fractures. D 2004 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Fracture; Menatetrenone; Vitamin D; Vitamin K

Introduction Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by progressive loss of memory and cognitive function. Also, far advanced AD is associated with generalized weakness. A high incidence of fractures, particularly of the hip [1–3], represents an important * Corresponding author. Department of Neurology, Mitate Hospital, 3237 Yugeta, Tagawa 826-0041, Japan. Fax: +81 947 46 3090. E-mail address: [email protected] (Y. Sato). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.09.018

problem in AD patients, who are prone to falls [4] and may have osteoporosis. The odds ratio of 6.9 for fracture prevalence between elderly persons with and without AD is reported [4]. In addition, functional recovery after hip fracture in AD is poor [5–7], and patients with dementia have increased mortality during the 6 months after hip fracture [8]. The physical state of AD patients has increasingly become one of the critical issues in the management of such patients. Our previous study [9] demonstrated that deficiency of 25hydroxyvitamin D (25-OHD) due to sunlight deprivation

62

Y. Sato et al. / Bone 36 (2005) 61–68

contributes to the reduced bone mineral density (BMD) in AD patients in nursing homes. Kipen et al. [10] examined demented women in the community and found that they have normal bone density, hypovitaminosis D, and compensatory hyperparathyroidism. Vitamin K is responsible for the site-specific carboxylation of osteocalcin (OC) and other bone matrix proteins [11]; and increased serum concentration of Glu OC is an indicator of compromised vitamin K status [12]. Increased serum Glu OC is associated with reduced BMD in the hip and a risk of fracture in otherwise healthy elderly women [13–16]. Nutritional vitamin K1 deficiency may reduce production of fully carboxylated OC, which may cause reduced BMD in elderly female patients with AD [17]. Low dietary vitamin K1 intake was associated with low BMD in women leading to an increased risk of hip fracture [18], and treatment with vitamin K2 (menatetrenone; MK-4) increases metacarpal BMD in senile osteoporosis [19] and reduces the incidence of fractures in patients with postmenopausal osteoporosis [20]. Thus, both vitamin D and vitamin K deficiencies contribute to the reduced BMD in AD patients. We therefore conducted a 2-year randomized open label trial to evaluate the efficacy of combined therapy with MK-4 plus vitamin D2 and calcium supplements in reducing the severity of the osteoporosis and decreasing the risk of nonvertebral fractures in elderly female patients with AD. The rate of vertebral fractures was not determined in this study because many vertebral fractures are asymptomatic among elderly AD patients and the interpretation of spinal X-ray films may be complicated by osteoarthritis or scoliosis.

Materials and methods We thought a trial size of 200 was appropriate due to our previous experience in a cohort study on hip fractures in AD patients. We recruited 200 ambulatory women, from the outpatient clinic of the Futase Social Insurance Hospital, who were over 70 years old and met DSM-III R criteria for probable AD [21]. Patients with impairment of renal, cardiac, or thyroid function or those who had known causes of osteoporosis, such as primary hyperparathyroidism or renal osteodystrophy, were excluded from this study. Patients were excluded if they had been treated with corticosteroids, estrogens, calcitonin, bisphosphonate, calcium, or vitamins D and K for 3 months or more during the 12 months preceding the study; and those who had been administered these agents for even a brief period during the preceding 3 months were also excluded. Totally disabled AD patients were excluded because their conditions virtually predicted minimum chance of fracture. Patients with a previous history of nonvertebral fracture were also excluded. At baseline, we assessed body mass index (BMI) and illness duration. The Mini-Mental State Examination (MMSE) [22] was given to the patients, and activities of

daily living (ADL) was assessed by the Rankin scale, in which a score of 0 indicates an independent daily living and a score of 5 indicates severe disability with total dependence that requires constant nursing care [23]. Mean weekly intake of dietary calcium and vitamins D and K1 (phylloquinone) during the previous 12 months was calculated for each individual from a questionnaire filled by patients or family members. Daily dietary intake of phylloquinone was assessed by a 126-item semiquantitative food frequency questionnaire (FFQ) [24,25]. Sunlight exposure during the preceding year was assessed by the patients or family members and graded as almost none, less than 15 min per week, or longer [26]. Falls were defined as incidents where the subject fell due to an unexpected loss of balance; patients who fell at least once in the 3 months before recruitment were defined as bfallers.Q The number of falls per a subject was also recorded during the 2-year follow-up period. As controls, cognitively normal age-matched women without fractures at any sites were recruited from the community. Control subjects were excluded if they had known causes of osteoporosis such as hyperparathyroidism; impairment of renal (serum creatinine N 1.5 mg/dl), cardiac, or thyroid function; a history of therapy with corticosteroids, estrogens, calcitonin, bisphosphonate, calcium, or vitamins D and K for 3 months or longer during the 12 months preceding the study; or even brief treatment with these agents during the 2 months immediately preceding the study. A total of 100 elderly women were followed as controls. All patients and controls were informed of the nature of the study. Consent was obtained from each participant, or from family members when patients were unable to understand because of dementia. The study protocol was approved by the local ethics committee. The AD patients were assigned, by means of computerassisted random numbering, either to the treated group with MK-4, ergocalciferol, and calcium (n = 100) or the untreated group (n = 100). MK-4 at a dose of 45 mg/day, ergocalciferol at a dose of 1000 IU/day, and elemental calcium at a dose of 600 mg/day were given to the treated group. The untreated group did not receive any medications. No placebo capsules were administered. No dose adjustments were made at any time during the study. The patients were prohibited from taking any other drugs that could affect bone and calcium metabolism. Follow-up assessment of patients’ condition was performed by physicians who did not participate in the initial randomization. The two patient groups were observed for 2 years. To confirm the effect of combined therapy upon BMD and fracture incidence, patients were divided into three groups according to their serum 25-hydroxyvitamin D (25-OHD) levels: severely deficient group (25-OHD b 5 ng/ml), deficient group (5 ng/ml V 25-OHD b 12 ng/ml), and insufficient group (12 ng/ml V 25-OHD b 20 ng/ml). A general medical evaluation, metacarpal BMD measurements, and laboratory values were assessed upon entry into the study to obtain baseline values, and again after 1 and 2 years. The patients’ clinical

Y. Sato et al. / Bone 36 (2005) 61–68

status was assessed at baseline and all of them were followed up every 4 weeks in the outpatient clinic; and nonvertebral fractures, if any, were recorded. Monthly blood samples were obtained from the patients to monitor the possible adverse effects of MK-4, such as liver and renal dysfunction. Ten patients in the treated group and 12 in the untreated group dropped out or withdrew from the study due to noncompliance, loss to follow-up, intercurrent illness, or death. Thus, a total of 178 patients (90 patients in the treated group and 88 in the untreated group) completed the trial. To discriminate BMD variations related to the treatment from those related to methods of measurement, a control group was followed concurrently. Control subjects visited the clinics 1 and 2 years after the enrollment, and nine of them dropped out. Thus, 91 control subjects completed the trial. The data, at the start, 1, and 2 years later, of the patients and controls who completed the cohort were analyzed. Medical and bone evaluations performed before randomization were used as the baseline values. The BMD of the right second metacarpal bone was measured, using a computed X-ray densitometer (CXD; Teijin Limited, Tokyo, Japan) [27], on the day of study entry and 12 and 24 months later. The CXD method measures bone density and cortical thickness at the middle of the second metacarpal bone, using a radiogram of the hand and an aluminum step wedge as a standard (20 steps; 1 mm/step). The computer calculated BMD on the basis of the pattern expressed as gradations along the aluminum step wedge. BMD was expressed as the thickness of an aluminum equivalent (mm Al) showing corresponding X-ray absorption. Precision errors (coefficients of variation) in measuring BMD by CXD have ranged from 0.2% to 1.2% [27]. On the day of bone evaluation, a fasting blood sample was obtained from the patients and controls. Blood samples were analyzed for vitamins K1 and K2 (menaquinone-4), intact parathyroid hormone (PTH), ionized calcium, GluOC, and 25-OHD. Serum concentrations of vitamin K including K1 (phylloquinone) and K2 were measured by high-performance liquid chromatography according to the method of Langenberg and Tjaden [28]. When vitamin K2 was undetectable, attempts were made to detect this form of the vitamin in concentrated serum. Serum 25-OHD was determined using a competitive protein binding assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). Ionized calcium was measured in freshly prepared serum collected under anaerobic conditions. An ion-selective electrode was used as part of an ionized calcium analysis system (NOVA Biochemical, Newton, MA). Intact PTH was measured by immunoradiometric assay (Nichols Institute Diagnostics). To measure Glu OC, a commercially available enzyme-immunoassay kit was used (Takara Shuzo Co., Shiga, Japan) [29]. Urinary deoxypyridinoline (D-Pyr) was measured with an enzyme immunoassay kit (Metra Biosystems Inc., CA). Urinary D-Pyr was expressed relative to the urinary creatinine concentration (Amol/mol creatinine)

63

[30]. These analyses were carried out in the Special Reference Hormone Reference Laboratory in Tokyo. All data obtained were withheld to all authors until completion of the study period in order to avoid bias. All statistical procedures were performed using Statview J 5.0 and SuperANOVA 1.11 software packages (Abacus Concepts, Berkeley, CA). Values are given as the mean F SD unless otherwise indicated. One-way ANOVA and Fisher’s protected least significant difference were used to assess baseline differences among the three groups. The unpaired t test was used to determine the significance of any difference between the two groups of patients with AD. Baseline differences of categorical data were tested by chi-square analysis. Spearman’s rank correlation coefficients (SRCC) were calculated to examine possible correlations between BMD and various parameters such as vitamin K, Glu OC, 25-OHD, and ionized calcium concentrations, or between PTH and 25-OHD. The primary end point was defined as the incidence of a nonvertebral fracture. The BMD measurements and the laboratory values were computed and expressed as a percentage change from the baseline. Then the two patient groups were compared with respect to their laboratory values by using the Wilcoxon rank sum test. All the three groups were compared with respect to BMD by analysis of covariance (ANCOVA). The difference in the incidence of fracture between the two patient groups or three patient groups defined by 25-OHD during the 2-year follow-up was tested by chi-square test. P values less than 5% were considered statistically significant.

Results Baseline characteristics of study subjects (Tables 1 and 2) Demographic and baseline clinical features of study patients are presented in Tables 1 and 2. There were no significant differences between the two patient groups in duration of illness, MMSE, Rankin scale, socioeconomic status, BMI, percentage of fallers, sunlight exposure, dietary intake of calcium and vitamins D and K1, BMD, and serum indices of bone metabolism. BMI was significantly lower in the both patient groups as compared to the control subjects. The average daily intakes of calcium and vitamin D and vitamin K1 in both groups of AD patients were significantly lower than those in control subjects. In addition, most of the patients in both groups had been exposed to sunlight less than 15 min a week because of reduced mobility, which is deduced from a mean Rankin scale of 2.0. Metacarpal BMD in the two patients groups was significantly lower than the control value. In the two patient groups, the baseline values of serum ionized calcium, vitamin K1, 25-OHD concentrations were low, while serum vitamin K2 levels were normal as compared to the controls. Serum Glu OC levels and urinary concentrations of D-Pyr were higher in both groups.

64

Y. Sato et al. / Bone 36 (2005) 61–68

Table 1 Demographic and baseline clinical characteristics of the female patients with Alzheimer’s disease at study entry P valuea

Characteristic

Control (n = 100)

Untreated group (n = 100)

Treated group (n = 100)

Age (years) Duration of illness* (years) Mini-Mental State Examinationc b0.0001 Interval since menopause (years) Rankin scaled Body mass index (kg/m2) Faller (%) Sunlight exposure/week N15 min b15 min Dietary intake of calcium (mg/day) Dietary intake of vitamin D (IU/day) Dietary intake of vitamin K1 (Ag/day)

78.0 F 6.1 – 28 F 4 21.1 F 8.0 – 21.8 F 2.3 –

78.1 F 5.9 6.0 F 3.1* 16 F 5* 21.0 F 7.6 2.0 F 1.3 19.2 F 1.7* 33 (33%)

78.1 F 5.3 6.0 F 3.6 16 F 5* 20.6 F 6.6 2.0 F 1.4 19.5 F 2.3* 29 (29%)

0.90 0.83b b0.0001 0.54e

82 (82%) 18 (18%) 1019 F 103 114 F 20 247 F 102

21 (21%) 79 (79%) 854 F 177* 81 F 29* 103 F 25*

19 (19%) 81 (81%) 862 F 151* 80 F 28* 102 F 24*

0.74e b0.0001 b0.0001 b0.0001

0.99 0.94b

Values are mean F SD. a Analysis of variance (ANOVA). b Unpaired t test. c The mean standard deviation of Mini-Mental Examination scores in cognitively normal subject (mean age 80.3 years, 70% women) has been reported as 28.5 F 1.4. d ADL was evaluated by the modified Rankin scale [23]. e Chi-squared analysis between treated and untreated groups. * P b 0.0001 vs. control group.

When the both patient groups were analyzed together, the BMD correlated positively with MMSE (r = 0.264, P = 0.0002), BMI (r = 0.152, P = 0.0321) and vitamin K1 (r = 0.175, P = 0.0138), and 25-OHD (r = 0.256, P = 0.0003) concentrations, while BMD correlated negatively with Rankin scale (r = 0.237, P = 0.0008), PTH (r = 0.193, P = 0.0065), and Glu OC (r = 0.191, P = 0.0069). There were negative correlations between serum 25-OHD and PTH (r = 0.313, P b 0.0001) suggesting the existence of compensatory hyperparathyroidism [9,10]. Bone changes and serum biochemical markers Fig. 1 shows the percent changes from baseline in the metacarpal BMD in the two patient groups and control subjects during the 2 years. The percent changes in BMD were +2.3 F 0.4 in the treated group, 5.2 F 0.7 in the untreated group, and 2.1 F 0.3 in the control group. The differences among the three groups were statistically

significant (treated group vs. untreated group, P b 0.0001; treated group vs. control group, P b 0.0001; untreated group vs. control group, P = 0.0006; based on ANCOVA). The percent changes, from baseline, in BMD are shown in Fig. 2. In the treated group (Fig. 2A), the average percent changes in BMD in three 25-OHD subgroups were +1.2 F 0.4 in the severely deficient group (25-OHD b 5 ng/ml, n = 22), +1.6 F 0.3 in the deficient group (25-OHDb12 ng/ml, n = 44), and +3.3 F 0.6 in the insufficient group (25-OHD b 20 ng/ml, n = 24). The insufficient group had significantly larger increase in BMD as compared to the severely deficient and deficient groups (P = 0.0033 and 0.0095, respectively; based on ANCOVA). Both the severely deficient and deficient groups benefited almost equally from the combined therapy and this may have been due to vitamin K supplementation. Fig. 2B shows the changes in BMD in the three untreated groups during the 2 years. The percent changes in BMD were 5.8 F 0.8 in the severely deficient group (25-OHD b 5 ng/ml, n = 23), 5.1 F 0.6

Table 2 Bone mineral density and various biochemical tests of control subjects and two groups of female patients with Alzheimer’s disease at baseline Control Bone mineral density (mm Al) Ionized calcium (mEq/L) Intact PTH (pg/ml) Vitamin K1 (ng/ml) Vitamin K2 (ng/ml) Glu OC (ng/ml) Deoxypyridinoline (Amol/mol creatinine) 25-OHD (ng/ml)

2.13 2.51 49 0.24 0.067 9.3 6.1 24.5

F F F F F F F F

0.24 0.12 14 0.16 0.077 3.9 2.1 6.0

Untreated group

Treated group

P valuea

1.89 F 0.32* 2.40 F 0.14* 79 F 17* 0.12 F 0.11* 0.066 F 0.058 14.5 F 4.5* 11.8 F 2.8* 9.2 F 3.5*

1.88 F 0.29* 2.40 F 0.13* 79 F 19* 0.12 F 0.11* 0.067 F 0.071 14.4 F 3.2* 11.7 F 2.6* 9.4 F 3.1*

b0.0001 b0.0001 b0.0001 b0.0001 0.99 b0.0001 b0.0001 b0.0001

Values are mean F SD. PTH, parathyroid hormone; Glu OC, Glu osteocalcin; 25-OHD, 25-hydroxyvitamin D. a Analysis of variance (ANOVA). * P b 0.0001 vs. control group.

Y. Sato et al. / Bone 36 (2005) 61–68

65

Fig. 1. Percent changes (mean F SE) from baseline in metacarpal bone mineral density (BMD) after 1 and 2 years in the two groups of patients and controls. During the 2 years, the differences in the percent changes in the BMD among the three groups were statistically significant (based on ANCOVA; treated group vs. untreated group, P b 0.0001; treated group vs. control group, P b 0.0001; untreated group vs. control group, P = 0.0006). Numbers in parentheses are the numbers of the subjects followed.

in the deficient group (25-OHD b 12 ng/ml, n = 42), and 2.7 F 0.5 in the insufficient group (25-OHD b 20 ng/ml, n = 23). The decrease in the insufficient group was significantly smaller as compared to both the severely deficient and deficient groups (P = 0.0035 and 0.0024, respectively; based on ANCOVA). Changes in various parameters during the 2-year study period are summarized in Table 3. MK-4 levels increased significantly in the treated group but not in the untreated group. There were no changes in the levels of vitamin K1 in the two groups. Serum levels of PTH, Glu OC, and D-Pyr decreased while ionized calcium and 25-OHD levels increased significantly in the treated group. Fracture incidence Fractures occurred in 22 of 88 patients in the untreated group during the 2-year study period (15 with hip fractures; two fractures each at the distal forearm and proximal femur; one each at the proximal humerus, ribs, and pelvis). Among the treated group, three fractures (two with hip fractures; one at the proximal femur) were observed. There were 86% fewer all nonvertebral fractures (3/90 vs. 22/88, P = 0.0003) in the treated group as compared to the untreated group. The odds ratio for all nonvertebral fractures in the untreated and treated group was 7.5 (95% confidence interval, 5.6–10.1). The number of all nonvertebral fractures per 1000 patientyears was 17 and 125 for the treated and untreated groups, respectively. There were 87% fewer hip fractures (2/90 vs.

Fig. 2. Percent changes (mean F SE) from baseline in metacarpal bone mineral density (BMD) after 1 and 2 years in the three treated groups (A) and in the three untreated groups (B) divided by 25-hydroxyvitamin D (25OHD) levels. In the treated group, the increase of BMD during the 2 years in both severe deficient (25-OHD b 5 ng/ml) and deficient groups (25OHD b 12 ng/ml) was similar (severe deficient group vs. deficient group, P = 0.30; severe deficient group vs. insufficient group, P = 0.0033; deficient group vs. insufficient group, P = 0.0095; based on ANCOVA). In the untreated group, the decrease of BMD during the 2 years in both severe deficient and deficient groups was similar and significantly smaller than in insufficient group (severe deficient group vs. deficient group, P = 0.24; severe deficient group vs. insufficient group, P = 0.0035; deficient group vs. insufficient group, P = 0.0024; based on ANCOVA). Numbers in parentheses are the numbers of the subjects followed.

66

Y. Sato et al. / Bone 36 (2005) 61–68

Table 3 Selected changes after 1 and 2 years in the 178 subjects who completed the study % change after 1 year (values at 1 year), P value* Ionized calcium (mEq/L) Untreated group Treated group Intact PTH (pg/ml) Untreated group Treated group Vitamin K1 (ng/ml) Untreated group Treated group Vitamin K2 (ng/ml) Untreated group Treated group Glu OC (ng/ml) Untreated group Treated group Deoxypyridinoline (Amol/mol creatinine) Untreated group Treated group 25-OHD (ng/ml) Untreated group Treated group

1.2 F 1.0 (2.34 F 0.01) 2.4 F 0.1 (2.34 F 0.01), b0.0001 +6.3 F 2.9 (81.0 F 17.8) 32.3 F 14.4 (52.4 F 14.6), b0.0001 5.3 F 4.7 (0.11 F 0.10) 4.8 F 4.5 (0.12 F 0.10), 0.77

% change after 2 year (values at 2 years), P value* 2.4 F 0.1 (2.30 F 0.01) +2.5 F 0.3 (2.49 F 0.09), b0.0001 +11.4 F 3.7 (86.9 F 21.3) 33.0 F 15.4 (51.1 F 12.1), b0.0001 8.9 F 5.6 (0.10 F 0.010) 8.7 F 5.1 (0.12 F 0.11), 0.44

1.8 F 1.0 (0.065 F 0.058) +278.0 F 23.3 (13.4 F 4.2), b0.0001

0.5 F 1.1 (0.066 F 0.059) +284.9 F 22.9 (14.4 F 4.8), b0.0001

+10.0 F 5.6 (15.4 F 4.6) 42.8 F 5.3 (8.2 F 1.9), b0.0001

+21.8 F 11.0 (17.5 F 5.0) 42.1 F 7.4 (8.3 F 2.0), b0.0001

+8.6 F 13.6 (12.8 F 3.3) 29.3 F 7.5 (8.2 F 1.8), b0.0001

+23.3 F 17.5 (14.5 F 3.7) 28.9 F 16.4 (8.3 F 2.6), b0.0001

4.9 F 6.3 (8.6 F 5.2) +146.2 F 88.0 (21.1 F 3.1), b0.0001

24.3 F 6.6 (7.0 F 5.5) +147.9 F 77.4 (21.3 F 3.2), b0.0001

Values are the mean F SEM. Abbreviations are as in Table 2. * Wilcoxon rank sum test.

15/88, P = 0.0022) in the treated group as compared to the untreated group. The odds ratio for hip fractures in the untreated group and treated group was 7.7 (95% confidence interval, 5.0–11.7). The number of hip fracture per 1000 patient-years was 11 and 85 for the treated group and untreated group, respectively. There was no significant difference between the two groups in the number of falls per subject during the 2 years (2.3 F 1.9 in the untreated group and 2.4 F 1.8 in the treated group). Among the subjects with severely deficient 25-OHD (b5 ng/ml), one hip fracture in the treated group and 10 fractures in the untreated group (seven with hip fractures; one fracture each at the distal forearm, proximal humerus, and ribs) were observed. There was a significant difference in the incidence of nonvertebral fractures between the two groups: 1/22 vs. 10/23 (P = 0.0188), and the odds ratio for fractures in the untreated and treated group was 9.6 (95% confidence interval, 4.5–20.7). Among the deficient group (25-OHD b 12 ng/ml), 1 hip fracture in the treated group and 9 fractures in the untreated group (seven hip fractures and one each in the distal forearm and proximal humerus) were observed; and the incidence of all nonvertebral fractures was significantly different between the two groups: 1/43 vs. 9/42 (P = 0.0183). The odds ratio for nonvertebral fractures in the two groups was 9.2 (95% confidence interval, 4.7–19.1). Among the insufficient group (25-OHD b 20 ng/ml), one proximal femur fracture in the treated group and three fractures in the untreated group (the hip, proximal femur, and pelvis) were observed. There was no significant difference in the all fracture incidence between the two groups: 1/22 vs. 3/23 (P = 0.61).

Adverse effects Three patients in the treated group experienced gastrointestinal symptoms such as epigastric discomfort and nausea, but they subsided within a week without discontinuing menatetrenone or ergocalciferol. No patient in the treated group experienced liver or renal dysfunction.

Discussion Metacarpal CXD measurement has been validated and its accuracy was found comparable to the better-known but lessavailable method of dual energy X-ray absorptiometry. In previous studies on patients with Parkinson’s disease (PD) or stroke, we found second metacarpal BMD by CXD to correlate with risk of hip fracture [31–33]. Therefore, reduced second metacarpal BMD in AD patients appears to reflect a decrease throughout the appendicular skeleton [34]. Among the elderly female patients with AD who completed the 2-year study, the numbers of hip fractures and all nonvertebral fractures in the treated group was lower than the untreated group by 87% and 86%, respectively. The incidence of all nonvertebral fractures during the 2 years in the untreated group was as high as 7.5, indicating a fracture rate of 125 per 1000 patient-years. Serum levels of vitamin D, vitamin K1, Glu OC, and PTH, immobilization (Rankin scale), BMI, and MMSE were found to correlate with BMD in elderly female patients with AD.

Y. Sato et al. / Bone 36 (2005) 61–68

The present study was confined to elderly women with AD, which explains the higher incidence of fractures as compared to the previous data obtained in the general population [35,36]. The high incidence of nonvertebral fractures particularly in the hip in elderly female patients with AD may be attributable to frequent falls and osteoporosis due to deficiency of vitamins D and K1, compensatory hyperparathyroidism, and immobilization. Since the number of falls was similar in the two groups during the observation period, MK-4 plus vitamin D2 and calcium supplements may have prevented nonvertebral fractures in AD patients despite frequent falls. The loss of BMD in the femoral neck, spine, and total body in untreated, community dwelling elderly patients of both genders has been reported to be less than 1% over 3 years [36]. In the present study, we found more pronounced bone loss in elderly female patients with AD. Over 2 years, the BMD decreased by 5.2% in the untreated patients and 2.1% in the control group, while the BMD increased by 2.3% in the treated group. The hip fracture rate in the untreated group was calculated as 85 per 1000 patient-years. The rate of hip fracture in an elderly reference population between 70 and 79 years old is reported to be 3.0 per 1000 patient-years [36]. Although the mean age of our AD subjects (78 years) was within this range, with no subjects younger than 70 or older than 88, the fracture rate in the present series was far higher than that reported in the reference population [36]. Vitamin K has two main sources in humans [37]: vitamin K1 is supplied by the diet, especially green leafy vegetables, while vitamin K2 is synthesized by bacteria in the gut. In the present study, a significant decrease in serum vitamin K1, but not K2, was observed in AD patients. Vitamin K1 deficiency in these patients was considered to reflect generally poor nutrition as also evidenced by low serum 25-OHD and lower BMIs. It has been reported that demented patients had malnutrition and decreased body weight [38]. In addition, sunlight deprivation may have contributed to 25-OHD deficiency. In our previous study on stroke patients [39], with both vitamins D and K1 deficiency, treatment with MK-4 for 1 year resulted in an increase in second metacarpal BMD by 4.3%, and the BMD in untreated controls decreased by 4.7%. Similar results were obtained by MK-4 therapy in PD patients [12], and the odds ratio of nonvertebral fractures in the untreated controls and the MK-4 group was 11.5 [12]. In the present study, 25-OHD deficiency with compensatory hyperparathyroidism resulted in high urinary excretion of D-Pyr. Treatment with ergocalciferol brought about increase in 25-OHD with increase in calcium and decrease in PTH and D-Pyr. Treatment with ergocalciferol increases serum 25-OHD concentrations [40–42], and ergocalciferol prevents fracture [43,44]. The present study was designed as a cohort to elucidate the effectiveness of MK-4, ergocalciferol, and calcium, and many cases in the untreated group with vitamin D deficiency were followed without vitamin D

67

supplementation since we were blind to serum 25-OHD levels before completion of the study. Both the patients with severely deficient and deficient 25-OHD levels in the treated group almost equally benefited from the therapy in terms of both increased BMD and prevention of nonvertebral fractures. This may imply that supplementation of MK-4 may be particularly effective in patients with marked vitamin D deficiency. A study has demonstrated that daily supplementation combining cholecalciferol (800 IU) with calcium (1200 mg) can reduce hip fracture by 43% in postmenopausal women [45]. Although cholecalciferol is not available in Japan, incidence of fracture may be more reduced by menatetrenone plus cholecalciferol and calcium supplements in elderly female patients with AD. We conclude that female AD patients with low serum vitamins D and K are at increased risk for nonvertebral fracture particularly in the hip. Combined treatment with MK-4, ergocalciferol, and calcium may be safe and effective in increasing bone mass and reducing the risk of fracture in elderly female patients with AD.

References [1] Johansson C, Skoog I. A population-based study on the association between dementia and hip fractures in 85-year olds. Aging (Milano) 1996;8:189 – 96. [2] Melton III LJ, Beard CM, Kokmen E, Atkinson EJ, O’Fallon WM. Fracture risk in patients with Alzheimer’s disease. J Am Geriatr Soc 1994;42:614 – 9. [3] van Staa TP, Leufkens HG, Cooper C. Utility of medical and drug history in fracture risk prediction among men and women. Bone 2000;31:508 – 14. [4] Buchner DM, Larson EB. Falls and fractures in patients with Alzheimer-type dementia. JAMA 1987;257:1492 – 5. [5] Holmes J, House A. Psychiatric illness predicts poor outcome after surgery for hip fracture: a prospective cohort study. Psychol Med 2000;30:921 – 9. [6] Matsueda M, Ishii Y. The relationship between dementia score and ambulatory level after hip fracture in the elderly. Am J Orthop 2000;29:691 – 3. [7] Morrison RS, Siu AL. Mortality from pneumonia and hip fractures in patients with advanced dementia. JAMA 2000;284:2447 – 8. [8] Nightingale S, Holmes J, Mason J, House A. Psychiatric illness and mortality after hip fracture. Lancet 2001;357:1264 – 5. [9] Sato Y, Asoh T, Oizumi K. High prevalence of vitamin D deficiency and reduced bone mass in elderly women with Alzheimer’s disease. Bone 1998;25:555 – 7. [10] Kipen E, Helme RD, Wark JD, Flicker L. Bone density, vitamin D nutrition, and parathyroid hormone levels in women with dementia. J Am Geriatr Soc 1995;43:1088 – 91. [11] Vermeer C, Jie KSG, Knapen MHJ. Role of vitamin K in bone metabolism. Annu Rev Nutr 1995;15:1 – 22. [12] Sato Y, Honda Y, Kaji M, Asoh T, Hosokawa K, Kondo I, et al. Amelioration of osteoporosis by menatetrenone in elderly female Parkinson’s disease patients with vitamin D deficiency. Bone 2002; 31:114 – 8. [13] Bitensky L, Hart JP, Catterrall A, Hodges SJ, Pilkington MJ, Chayen J. Circulating vitamin K levels in patients with fractures. J Bone Joint Surg 1988;70B:662 – 3. [14] Hart JP, Shearer MJ, Klenerman L, Catterall A, Reeve J, Sambrook PN,

68

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

[27]

[28]

[29]

Y. Sato et al. / Bone 36 (2005) 61–68 et al. Electrochemical detection of depressed circulating levels of vitamin K1 in osteoporosis. J Clin Endocrinol Metab 1985;60:1268 – 9. Hodges SJ, Akesson K, Vergnaud P, Obrant K, Delmas PD. Circulating levels of vitamin K1 and K2 decreased in elderly women with hip fractures. J Bone Miner Res 1993;8:1241 – 5. Hodges SJ, Pilkington MJ, Stamp TCB, Catterall A, Shearer MJ, Bitensky L, et al. Depressed levels of circulation menaquinones in patients with osteoporotic fractures of the spine and femoral neck. Bone 1991;12:387 – 9. Sato Y, Honda Y, Hayashida N, Iwamoto J, Kanoko T, Satoh K. Vitamin K deficiency and osteopenia in elderly female patients with Alzheimer’s disease. Arch Phys Med Rehabil [in press]. Booth SL, Broe KE, Gagnon DR, Tucker KL, Hannan MT, McLean RR, et al. Vitamin K intake and bone mineral density in women and men. Am J Clin Nutr 2003;77:512 – 6. Orimo H, Shiraki M, Tomita A, Morii H, Fujita T, Ohata M. Effects of menatetrenone on the bone and calcium metabolism in osteoporosis: a double-blind placebo-controlled study. J Bone Miner Metab 1998;16: 106 – 12. Szulc P, Arlot M, Chapuy MC, Meunier PJ, Delmas PD. Serum undercarboxylated osteocalcin correlates with hip bone mineral density in elderly women. J Bone Miner Res 1994;9:1591 – 5. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Standian EM. Clinical diagnosis of Alzheimer’s disease: report on the NINCDS-ADRADA work group under the auspices of the Department of Health and Human Services Task Force on Alzheimer’s disease. Neurology 1984;34:939 – 44. Folstein MF, Folstein SF, McHugh PR. bMini Mental State.Q A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12:189 – 98. Van Swieten JC, Koudstaal PJ, Visser MC. Interobserver agreement for assessment of handicap in stroke patients. Stroke 1988;19:604 – 7. Rimm EB, Giovannucci EL, Stampfer MJ, Colditz GA, Litin LB, Willett WC. Reproducibility and validity of an expanded selfadministered semiquantitative food frequency questionnaire among male health professionals. Am J Epidemiol 1992;135:1114 – 26. Feskanich D, Weber P, Willett WC, Rockett H, Booth SL, Colditz GA. Vitamin K intake and hip fractures in women: a prospective study. Am J Clin Nutr 1999;69:74 – 9. Komar L, Nieves J, Cosman F, Rubin A, Shen V, Lindsay R. Calcium homeostasis of an elderly population upon admission to a nursing home. J Am Geriatr Soc 1993;41:1057 – 64. Matsumoto C, Kushida K, Yamazaki K, Imose K, Inoue T. Metacarpal bone mass in normal and osteoporotic Japanese women using computed X-ray densitometry. Calcif Tissue Int 1994;55:324 – 9. Langenberg JP, Tjaden UR. Improved method for the determination of vitamin K1 epoxide in human plasma with electrofluorimetric reaction detection. J Chromatogr 1984;289:377 – 85. Vergnaud P, Garnero P, Meunier PJ, Breart G, Kamihagi K, Delmas PD. Undercarboxylated osteocalcin measured with a specific immuno-

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37] [38]

[39]

[40]

[41]

[42]

[43] [44]

[45]

assay predicts hip fracture in elderly women: the EPIDOS Study. J Clin Endocrinol Metab 1997;82:719 – 24. Robins SP, Black D, Petersen CR. Evaluation of urinary hydroxypyridinium cross-link measurements as resorption markers in metabolic bone disease. Eur J Clin Invest 1991;21:310 – 5. Sato Y, Manabe S, Kuno H, Oizumi K. Amelioration of osteopenia and hypovitaminosis D by 1a-hydroxyvitamin D3 in Parkinson’s disease. J Neurol, Neurosurg Psychiatry 1999;66:64 – 8. Sato Y, Maruoka H, Oizumi K. Amelioration of hemiplegia-associated osteopenia over 4 years following stroke by 1 a-hydroxyvitamin D3 and calcium supplementation. Stroke 1997;28:736 – 9. Sato Y, Kuno H, Kaji M, Saruwatari N, Oizumi K. Effect of ipriflavone on bone in elderly hemiplegic stroke patients with hypovitaminosis D. Am J Phys Med Rehabil 1999;78:457 – 63. Derisquebourg T, Dubois P, Devogelaer JP, Meys E, Duquesnoy B, Nagant de Deuxchaisnes C, et al. Automated computerized radiogrammetry of the second metacarpal and its correlation with absorptiometry of the forearm and spine. Calcif Tissue Int 1994;54:461 – 5. Dawson-Hughes B, Harris SS, Krall EA, Dallal GE. Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older. N Engl J Med 1997;337:670 – 6. Ramnemark A, Nyberg L, Borsse´n B, Olsson T, Gustafson Y. Fractures after stroke. Osteoporos Int 1998;8:92 – 5. Shearer MJ. Vitamin K. Lancet 1995;345:229 – 34. Burns A, Marsh A, Bender DA. Dietary intake and clinical, anthropometric and biochemical indices of malnutrition in elderly demented patients and non-demented subjects. Psychol Med 1989;19:383 – 91. Sato Y, Honda Y, Kuno H, Oizumi K. Menatetrenone ameliorates osteopenia in disuse-affected limbs of vitamin D- and K-deficient stroke patients. Bone 1998;23:291 – 6. Francis RM, Boyle IT, Moniz C, Sutcliffe AM, Davis BS, Beastall GH, et al. A comparison of the effects of alfacalcidol treatment and vitamin D2 supplementation on calcium absorption in elderly women with vertebral fractures. Osteoporos Int 1996;6:284 – 90. Lark RK, Lester GE, Ontjes DA, Blackwood AD, Hollis BW, Hensler MM, et al. Diminished and erratic absorption of ergocalciferol in adult cystic fibrosis patients. Am J Clin Nutr 2001;73:602 – 6. Sambrook PN, Kotowicz M, Nash P, Styles CB, Naganathan V, Henderson-Briffa KN, et al. Prevention and treatment of glucocorticoid-induced osteoporosis: a comparison of calcitriol, vitamin D plus calcium, and alendronate plus calcium. J Bone Miner Res 2003;18: 919 – 24. Guirguis-Blake J, Phillips RL. Oral vitamin D3 decreases fracture risk in the elderly. J Fam Pract 2003;52:431 – 5. Heikinheimo RJ, Inkovaara JA, Harju EJ, Haavisto MV, Kaarela RH, Kataja JM, et al. Annual injection of vitamin D and fractures of aged bones. Calcif Tissue Int 1992;51:105 – 10. Chapuy MC, Arlot ME, Duboeuf FD, Brun J, Crouzet B, Arnaud S, et al. Vitamin D3 and calcium to prevent hip fractures in elderly women. N Engl J Med 1992;327:1637 – 42.