Cranberry juice improved antioxidant status without affecting bone quality in orchidectomized male rats.

Abstract

Background: We reported that drinking citrus juice improves bone quality in orchidectomized senescent male rats. Because cranberry juice, like citrus, is rich in nutrients and phenolic compounds, beneficial effects of citrus juice might also be seen with cranberry juice. An experiment evaluated effect of drinking cranberry juice on bone quality in orchidectomized rats.

Methods: Thirty-two 1-year-old male rats were randomized to two groups: a sham-control group (n = 8) and an orchidectomized group (n = 24). The treatments for the 4 months duration of the study were SHAM, orchidectomy (ORX), ORX + drinking either 27% or 45% cranberry juice concentrate added to drinking water. At the termination of the study, the rats were euthanized, blood was collected for plasma antioxidant status and IGF-I. The femur, tibia and the 4th lumbar were evaluated for bone quality. Total calcium and magnesium concentration in the femurs were also evaluated.

Results: ORX did not affect red blood cell (RBC)-induced hemolysis despite lowering (p < 0.05) plasma antioxidant capacity; reduced (p < 0.05) plasma IGF-I, femoral density, femoral strength, time-induced femoral fracture, bone mineral content, bone mineral area; numerically (p = 0.07) lowered 4th lumbar density; decreased (p < 0.05) trabecular connectivity, trabecular number, femoral ash; increased (p < 0.05) trabecular separation in comparison to the SHAM group. Drinking cranberry juice increased (p < 0.05) plasma antioxidant status, protected RBC against hemolysis, but had no positive effect on bone quality or bone mineral status.

Conclusions: Cranberry juice increases plasma antioxidant status without affecting bone quality.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Cranberry juice; Vaccinium macrocarpon; Bone quality; Rats; Antioxidant status

Introduction

Osteoporosis is a chronic disease of aging that is characterized by micro-architectural deterioration of bone that leads to bone fragility and increase the risk of fractures (National Institute of Health, 2006). Although osteoporosis mostly occurs in women, men also experience osteoporosis. Previously, we reported a decrease in bone quality in male senescent rats following orchidectomy (ORX) (Deyhim et al., 2006). In the same study, we observed that the decrease in bone quality in orchidectomized rats coincided with reduced plasma antioxidant status and that increased plasma antioxidant capacity resulted in improvement of bone quality (Deyhim et al., 2006). Reactive oxidative species (ROS) have been implicated in the development of osteoporosis. Oxidative stress plays a significant role in the pathophysiology of primary male osteoporosis by increasing bone resorption (Garrett et al., 1990; Yalin et al., 2005).

Osteoclasts have been reported to generate high levels of superoxide anion and hydrogen peroxide, which modulate intra- and intercellular signaling responsible for bone loss (Lean et al., 2005). In one animal study, vitamin C injection normalized castration-induced increase in the number of osteoclasts and prevented bone loss (Lean et al., 2003).

Fruits and vegetables are rich in vitamins, minerals, and bioactive compounds with antioxidant properties that can protect against development of chronic diseases (Negri et al., 1991; Steinmetz and Potter, 1991; Keli et al., 1996; Kris-Etherton et al., 2002; Ariga, 2004). The health-promoting effects of bioactive compounds have been the subjects of numerous studies (Poulose et al., 2005; Soobrattee et al., 2005; Yu et al., 2005; Lin et al., 2006).

Feeding hesperidin, has been shown to prevent bone loss (Chiba et al., 2003). In other studies, feeding onion and Italian parsley, which are rich in flavonoid have decreased bone resorption in rats (Muhlbauer and Li, 1999; Horcajada-Molteni et al., 2000) Cranberries contain flavonoids including proanthocyanidins, anthocyanins, and flavonols, which may protect bone against resorption. The antioxidant capacity of proanthocyanidin has been reported to be stronger than vitamin C, vitamin E, and catechins (Ariga, 2004). Previously, we reported that drinking citrus juice improves bone quality in orchidectomized senescent male rats (Deyhim et al., 2006). Because cranberry juice, like citrus, is rich in nutrients and phenolic compounds, beneficial effects of citrus juice might also be seen with cranberry juice. If the benefit of cranberry juice is related to its antioxidant capacity, drinking the juice might dose-dependently improve the micro-architecture of bone in orchidectomized rats. The objective of the present study was to evaluate the efficacy of drinking cranberry juice on improving bone quality in orchidectomized senescent male rats.

Materials and methods

Animals and diet

In this experiment, 32 retired male breeder rats upon arrival at Texas A & M University-Kingsville were housed individually in an environmentally controlled laboratory for 3 days of acclimation prior to either surgical ORX or sham surgery. The animals were weighed and divided in two groups: sham (control) group (n = 8) and orchidectomized group (n = 24). Three days post-surgery when feed intake was normalized, initial body weight was taken and orchidectomized rats were assigned to one of the following three treatments to be compared with the sham (control) group; ORX group drinking 27% cranberry juice concentrate diluted in deionized water, and ORX group drinking 45% cranberry juice concentrate diluted in deionized water. Commercial cranberry drink contains 27% cranberry juice concentrate. Total phenolic and anthocyanin content of cranberry juice is reported to be 1136 [+ or -] 3.5 (mg/l gallic acid equivalents) and 2.80 (mg/l malvidin-3-glycoside equivalents), respectively (Duthie et al., 2006). Guidelines for the ethical care and treatment of animals from the Animal Care and Use Committee of Texas A & M University-Kingsville were strictly followed. The animals ate semi-purified, powdered casein-based diet, AIN-93M, for the duration of 4 months study. Food consumption was monitored every 3 days and all ORX rats were pair-fed to the mean food intake of the sham group. Table sugar (10 [+ or -] 0.5 g, 42 kcal/100 ml) and baking soda (13 g/l) were added to cranberry juice to adjust for tartness and pH. Sham (control) and ORX groups received deionized water (DI) containing table sugar adjusted for calorie (10 [+ or -] 0.5g; 42 kcal/100 ml) and baking soda (13 g/l) was added for neutralizing pH to 7.2 [+ or -] 0.25. All rats were pair-fed to the mean food intake of the SHAM group.

Blood parameter

Four months after drinking cranberry juice, rats were euthanized and bled from the abdominal aorta. Blood samples were collected in heparinized tubes and centrifuged (4[degrees]C) at 1500g for 15min. Plasma was separated and an aliquot of plasma was refrigerated for oxidative stress-induced RBC hemolysis, an index for RBC resistance to hemolysis. The kinetics of RBC resistance to hemolysis was determined at 27[degrees]C by continuous monitoring of changes in 450-nm absorbance. The time to reach 50% of total hemolysis ([T.sub.50% hemolysis]) was used for group comparisons (Blache and Prost, 1992). Plasma antioxidant capacity was evaluated using a commercially available kit (Calbiochem, San Diego, CA) as a quantitative measure of circulating antioxidant status. Plasma IGF-I was evaluated using a commercially available kit (R & D Systems, Minneapolis, MN) as a quantitative measure of bone formation.

Bone quality assessment

The femur was cleaned of soft tissue and stored in a glass vial at -20[degrees]C. Femoral length was measured using a digital caliper. Femoral density analysis was evaluated using Archimedes' principle (Deyhim et al., 2003). Briefly, according to Archimedes' principle, a buoyant force on a submerged object in a fluid equals the density of the fluid multiplied by the volume of the fluid that is displaced. The latter is the ratio of buoyant force to fluid density. The density of the object is then obtained as the mass divided by the volume that is displaced. However, it is important to note that the buoyant force does not depend on the weight or shape of the submerged object but on the weight of the displaced fluid. Although assessing bone density using Archimedes' principle is time consuming but it measures true rather than apparent density.

Femoral biomechanical properties were measured by a three-point bending test (Stable Micro System, Canton, MA). The bone samples were placed in similar orientation on two rods spaced 20 mm apart and the force was delivered to the mid-shaft by a crosshead moving at a constant speed of 1.0 mm/min. A force versus deformation curve was recorded. Ultimate force provided a measure of bending strength of the intact femur (Kiebzak et al., 1988). The time to cause bone breakage was recorded by the texture analyzer (Stable Micro System, Canton, MA). The outer diameters of the bones at the point of fracture were measured with a micrometer to calculate cortical area assuming an elliptical cross-section.

Dual Energy X-ray Absorptiometry (DEXA)

Bone mineral area (BMA), bone mineral content (BMC), and bone mineral density (BMD) were evaluated via dual-energy X-ray absorptiometry using high-resolution software designed for analysis of small animal bones (DEXA; QDR-4500A Elite; Hologic Inc., Waltham, MA). Prior to scanning, the 4th lumbar vertebrae were placed in deionized water in standard orientation in plastic weighing boats. Similarly, tibias were scanned dry and were also placed in a consistent orientation.

Micro-computed tomography ([micro]CT) analysis of 4th lumbar vertebra and tibia

Micro-computed tomography ([micro]CT) imaging of 4th lumbar vertebrae and tibial metaphysis was performed with a [micro]CT 40 scanner (Scanco Medical AG, Zurich, Switzerland). Using an integration time of 150 ms per projection, scanning was completed using medium resolution with 55 kVP energy, 144 [micro]A current with an isotropic voxel resolution of 16 [micro]m. The operator was blinded to the treatment for the analyses. Bones were scanned in a 16 mm plastic tube from the proximal growth plate in the distal direction (16 [micro]m/slice). Approximately 400 (16 [micro]m/slice) slices resulted from scanning 4th lumbar vertebra from the caudal to the dorsal end. Total contoured slices for L-4 vertebra averaged 250 slices, with volume of interest (VOI) evaluations based on selected regions of trabecular bone beginning 10 slices from the growth plate at each end. VOI evaluations for tibias were also based on selected regions of secondary spongiosa beginning 35 slices from the growth plate bridge at the proximal end and continuing for 100 slices. Thirty slices were contoured to constitute the VOI for the mid-shaft evaluations of cortical bone.

Bone micro-architectural parameters were acquired through VOI evaluations. Parameters of interest focused on bone volume fraction (BV/TV), trabecular number (Tb. N.), trabecular separation (Tb. Sp.), trabecular thickness (Tb. Th.), connectivity density (Conn. D.), and cortical thickness (Cort. Th.). Lumbar vertebrae and tibia evaluations were segmented using a Gaussian filter (sigma 0.7, support 1) to remove noise and a fixed threshold level of 240 to obtain a 3-D bone structure. Tibial mid-shafts were analyzed using a fixed threshold level of 280.

Ash

Femurs from all treatment groups were placed in a drying oven at 100[degrees]C for 72 h. Once bone weight was stabilized, samples were weighed, pulverized, and ashed in covered crucibles at 600[degrees]C for 16h. Samples were cooled and weighed to determine the percent ash. The ashed samples were diluted with 4N HCl solution and an aliquot was taken and re-diluted with 0.5% nitric acid and 0.5% lanthanum chloride solution. Bone calcium and magnesium were analyzed using flame atomic absorption spectrophotometry (Model 800, Perkin-Elmer, Norwalk, CT) and air-acetylene flame.

Statistical analysis

Data were analyzed by analysis of variance using the General Linear Models procedure of SAS to determine the effect of drinking cranberry juice on antioxidant status, bone quality, and concentrations of calcium and magnesium in femur as the primary outcome variables. When a significant F statistic (p < 0.05) from analysis of variance was calculated, the least square means procedure was performed for separating means that were significantly (p < 0.05) different (SAS, 1999).

Results

In the present study, initial weight was not different between treatment groups (Table 1). Drinking cranberry juice for 4 months duration of this study did not (p > 0.1) affect final weight, drinking, or food consumption among groups (Table 1).

Although plasma antioxidant capacity decreased (p < 0.05) by orchidectomizing rats, RBC hemolysis percentage was not (p > 0.1) different in comparison to the sham group (Table 1). Drinking cranberry juice increased (p < 0.05) plasma antioxidant capacity and protected RBC against hemolysis in orchidectomized rats (Table 1).

As expected, ORX significantly (p < 0.05) decreased plasma IGF-I, femoral strength, and the time it took to cause femoral breakage (Table 1). Also, BMA, BMC, BV/TV, trabecular number, trabecular connectivity decreased (p < 0.05) and trabecular separation increased (p < 0.05) with ORX in comparison with the control (sham) group (Table 2). The 4th lumbar density tended to decrease (p = 0.07) while left tibia density numerically decreased with ORX in comparison with the sham group (Table 2). Irrespective of dosage, drinking cranberry juice had no (p > 0.1) beneficial effect on plasma IGF-I, femoral strength (Table 1), or overall bone quality (Table 2) in comparison with the ORX group.

In the present study, ORX decreased (p < 0.1) femoral length as well as total calcium, magnesium and ash in the femur (Table 1). Drinking cranberry juice did not affect calcium and magnesium in the femur in comparison with the ORX group (Table 1).

Discussion

As expected, food and water intake along with final body weight were similar across treatments because all rats were pair-fed to the mean food intake of the sham-control group. Adjusting the pH of drinking solutions resulted in drinking quantities of fluid that were not different across treatments.

Oxidative stress is an imbalance between generation of reactive oxygen species and the ability of cellular enzymatic and non-enzymatic antioxidant defense systems to detoxify free radicals (Halliwell and Gutteridge, 1989). The present results demonstrated that although ORX lowered antioxidant capacity, plasma antioxidant status in the ORX group was still adequate to protect RBC against free radical-induced hemolysis. In contrast, drinking cranberry juice increased plasma antioxidant capacity and improved RBC resistance to hemolysis. Drinking 27% cranberry juice improved RBC resistance slightly, but 45% cranberry juice protected RBC against pro-oxidant-induced hemolysis at a level exceeding that of the control (sham) group. Protective effects of cranberry juice may be associated to its bioactive content (Dembinski et al., 2004; Tirkey et al., 2005).

In this study, decreased plasma IGF-I in orchidectomized rats was indicative of slowed bone formation. Plasma concentration of IGF-I in rats drinking cranberry juice was not different from those of the ORX group suggesting that cranberry juice at the dosage given has no beneficial effect on bone formation.

The loss of the protective effect of testosterone on bone remodeling may have been the reason for increased fracture rate observed in the ORX group. Reduced femoral strength observed in the ORX group is related to reduced femoral density. Reduced bone growth and bone formation as evident from decreased plasma IGFI, femoral length, and trabecular number may have contributed to reduced total calcium, magnesium and ash in the femurs from the ORX group. However, we suggest that bone quality is an integration of bone composition and bone modeling. Certainly, BMC contributes to bone quality but our results clearly demonstrate that ORX also significantly impacted micro-architecture of tibia and the 4th lumbar vertebrae as shown by decreased BV/TV, Conn. D., Tb.N. and increased trabecular separation. These changes may be attributed to increased bone resorption and reduced bone formation. Although trabecular number and trabecular separation were significantly affected by ORX, it did not affect trabecular morphology as evident from trabecular thickness. Cranberry juice did not affect any of the bone variables evaluated in this study in comparison with the ORX group. Our results suggest that drinking cranberry juice did not have beneficial effect on bone quality of orchidectomized senescent male rats. However, we conclude that cranberry juice increased antioxidant capacity without affecting bone quality or bone mineral status.

Acknowledgments

This project is based upon work supported by the USDA CSREES IFAFS #2001-52102-02294 and USDA-CSREES #2005-34402-14401 "Designing Foods for Health" through the Vegetable & Fruit Improvement Center. The authors are grateful to Becky Bailey and Rori Morrow for assisting with analysis of the bone samples using DEXA and [micro]CT.

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A. Villarreal (a), B.J. Stoecker (b), C. Garcia (a), K. Garcia (a), R. Rios (a), C. Gonzales (a), K. Mandadi (c), B. Faraji (d), B.S. Patil (e), F. Deyhim (a,e,*)

(a) Department of Human Sciences, MSC 168, Texas A & M University-Kingsville, Kingsville, TX 78363, Kingsville, USA

(b) Nutritional Sciences, Oklahoma State University, Stillwater, OK 74078, USA

(c) Texas A & M University-Kingsville Citrus Center, Kingsville, TX 78596, USA

(d) The University of Texas-Pan American, Coordinated Program in Dietetics, Edinburg, TX 78541, USA

(e) Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A & M University, College Station, TX 77843, USA

* Corresponding author. Department of Human Sciences, MSC 168, Texas A & M University-Kingsville, Kingsville, TX 78363, Kingsville, USA. Tel.: + 1 361 593 2409; fax: + 1 361 593 2230.

E-mail address: [e-mail] (F. Deyhim).

Table 1. The effects of drinking cranberry juice for 4 months on
antioxidant status, femoral quality, and calcium and magnesium
concentrations in femur of orchidectomized rats

Variable            SHAM                       ORX

Initial body        468 [+ or -] 13            450 [+ or -] 10
  weight (g)
Final body          580 [+ or -] 11            573 [+ or -] 11
  weight (g)
Food intake (g)      22 [+ or -] 0.63           21.0 [+ or -] 0.37
Water intake (ml)    23 [+ or -] 3              26 [+ or -] 3

Plasma
Antioxidant           1.23 [+ or -] 0.04 (a)     1.09 [+ or -] 0.04 (b)
  capacity (mM)
RBC oxidative        85 [+ or -] 2 (b)          86 [+ or -] 2 (b)
  resistance (%)
IGF-1 (ng/ml)         3.20 [+ or -] 0.11 (a)     2.86 [+ or -] 0.10 (b)

Femur
Density (g/           1.486 [+ or -] 0.01 (a)    1.442 [+ or -] 0.04 (b)
  [cm.sup.3])
Cortical area (mm)   10.09 [+ or -] 0.27 (a)     7.39 [+ or -] 0.29 (b)
Femoral length        4.03 [+ or -] 0.02 (a)     3.81 [+ or -] 0.02 (b)
  (cm)
Femoral strength     30 [+ or -] 0.8 (a)        23 [+ or -] 0.7 (b)
  (kg)
Time induced bone     1.54 [+ or -] 0.05 (a)     1.27 [+ or -] 0.05 (b)
  fracture (s)
Ash (mg)            689 [+ or -] 18 (a)        609 [+ or -] 17 (b)
Calcium (mg)        277 [+ or -] 5 (a)         238 [+ or -] 5 (b)
Magnesium (mg)        5.56 [+ or -] 0.12 (a)     5.16 [+ or -] 0.11 (b)

                         ORX + cranberry juice
Variable            27%                        45%

Initial body        441 [+ or -] 12            451 [+ or -] 11
  weight (g)
Final body          596 [+ or -] 15            599 [+ or -] 13
  weight (g)
Food intake (g)      20.0 [+ or -] 0.49         21.0 [+ or -] 0.42
Water intake (ml)    24 [+ or -] 3              20 [+ or -] 3

Plasma
Antioxidant           1.24 [+ or -] 0.04 (a)     1.24 [+ or -] 0.04 (a)
  capacity (mM)
RBC oxidative        88 [+ or -] 2 (a,b)        94 [+ or -] 2 (a)
  resistance (%)
IGF-1 (ng/ml)         2.78 [+ or -] 0.14 (b)     2.73 [+ or -] 0.11 (b)

Femur
Density (g/           1.449 [+ or -] 0.01 (b)    1.446 [+ or -] 0.01 (b)
  [cm.sup.3])
Cortical area (mm)    8.65 [+ or -] 0.31 (b)     8.39 [+ or -] 0.31 (b)
Femoral length        3.84 [+ or -] 0.02 (b)     3.85 [+ or -] 0.02 (b)
  (cm)
Femoral strength     24 [+ or -] 0.7 (b)        24 [+ or -] 0.7 (b)
  (kg)
Time induced bone     1.34 [+ or -] 0.05 (b)     1.35 [+ or -] 0.05 (b)
  fracture (s)
Ash (mg)            606 [+ or -] 18 (b)        590 [+ or -] 19 (b)
Calcium (mg)        235 [+ or -] 5 (b)         226 [+ or -] 6 (b)
Magnesium (mg)        4.91 [+ or -] 0.12 (b)     4.96 [+ or -] 0.14 (b)

(a,b) Means with unlike superscripts within a row are significantly
different (p < 0.05).

Table 2. The effects of drinking cranberry juice for 4 months on bone
quality in 4th lumbar and tibia of orchidectomized rats

Variable            SHAM                       ORX

Lumbar (4th)
BMA ([cm.sup.2])     0.828 [+ or -] 0.033 (a)   0.725 [+ or -] 0.013 (b)
BMC (g)              0.230 [+ or -] 0.008 (a)   0.194 [+ or -] 0.006 (b)
BMD (g/[cm.sup.2])   0.278 [+ or -] 0.005       0.268 [+ or -] 0.008
Bone volume/total   23.2 [+ or -] 1.5 (a)      18.5 [+ or -] 1.3 (b)
  volume (%)
Connectivity        40.1 [+ or -] 3.8 (a)      27.4 [+ or -] 2.8 (b)
  density (1/
  [mm.sup.3])
Trabecular number    3.166 [+ or -] 0.165 (a)   2.512 [+ or -] 0.153 (b)
  (1/mm)
Trabecular           0.086 [+ or -] 0.002       0.086 [+ or -] 0.002
  thickness (mm)
Trabecular           0.312 [+ or -] 0.016 (b)   0.411 [+ or -] 0.029 (a)
  separation (mm)

Tibia
BMA ([cm.sup.2])     2.303 [+ or -] 0.052 (a)   2.063 [+ or -] 0.033 (b)
BMC (g)              0.599 [+ or -] 0.019 (a)   0.511 [+ or -] 0.014 (b)
BMD (g/[cm.sup.2])   0.259 [+ or -] 0.005       0.247 [+ or -] 0.004
Bone volume/total   16.2 [+ or -] 1.2 (a)      13.0 [+ or -] 0.8 (b)
  volume (%)
Connectivity        32.2 [+ or -] 3.0 (a)      22.0 [+ or -] 2.9 (b)
  density (1/
  [mm.sup.3])
Trabecular number    2.891 [+ or -] 0.169 (a)   2.338 [+ or -] 0.154 (b)
  (1/mm)
Trabecular           0.085 [+ or -] 0.005       0.087 [+ or -] 0.002
  thickness (mm)
Trabecular           0.344 [+ or -] 0.028 (a)   0.439 [+ or -] 0.036 (b)
  separation (mm)

                         ORX + cranberry juice
Variable            27%                        45%

Lumbar (4th)
BMA ([cm.sup.2])     0.715 [+ or -] 0.022 (b)   0.748 [+ or -] 0.03 (b)
BMC (g)              0.183 [+ or -] 0.010 (b)   0.199 [+ or -] 0.007 (b)
BMD (g/[cm.sup.2])   0.254 [+ or -] 0.006       0.266 [+ or -] 0.004
Bone volume/total   17.5 [+ or -] 1.5 (b)      17.4 [+ or -] 2.1 (b)
  volume (%)
Connectivity        24.6 [+ or -] 2.6 (b)      25.3 [+ or -] 3.1 (b)
  density (1/
  [mm.sup.3])
Trabecular number    2.368 [+ or -] 0.190 (b)   2.446 [+ or -] 0.187 (b)
  (1/mm)
Trabecular           0.085 [+ or -] 0.001       0.085 [+ or -] 0.003
  thickness (mm)
Trabecular           0.455 [+ or -] 0.047 (a)   0.422 [+ or -] 0.029 (a)
  separation (mm)

Tibia
BMA ([cm.sup.2])     2.082 [+ or -] 0.070 (b)   1.986 [+ or -] 0.052 (b)
BMC (g)              0.512 [+ or -] 0.025 (b)   0.489 [+ or -] 0.019 (b)
BMD (g/[cm.sup.2])   0.245 [+ or -] 0.005       0.245 [+ or -] 0.005
Bone volume/total   12.7 [+ or -] 0.6 (b)      11.2 [+ or -] 0.8 (b)
  volume (%)
Connectivity        18.8 [+ or -] 2.0 (b)      17.3 [+ or -] 1.9 (b)
  density (1/
  [mm.sup.3])
Trabecular number    2.178 [+ or -] 0.114 (b)   2.051 [+ or -] 0.121 (b)
  (1/mm)
Trabecular           0.088 [+ or -] 0.002       0.087 [+ or -] 0.003
  thickness (mm)
Trabecular           0.464 [+ or -] 0.029 (b)   0.497 [+ or -] 0.042 (b)
  separation (mm)

(a,b) Means with unlike superscripts within a row are significantly
different (p < 0.05).