International Journal of Cardiovascular ResearchISSN: 2324-8602

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Research Article, Int J Cardiovasc Res Vol: 4 Issue: 3

Aged Garlic Extract with Supplement is Associated with Beneficial Effect on Bone Mineral Density and Predicts Lack of Progression of Atherosclerosis: A Prospective Double Blinded Randomized Trial

Naser Ahmadi*, Vahid Nabavi, Hussein Zughaib, Nichole Patel, Avinash Rathod, Ferdinand Flores, Song Mao, Fereshteh Hajsadeghi and Matthew Budoff
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USA
Corresponding author : Naser Ahmadi, MD
David Geffen School of Medicine, UCLA, 1124 W. Carson Street, RB3 Torrance, CA, 90503, USA
Tel: + 310-803-0443; Fax: 310-222-5401
E-mail: [email protected]
Received: March 11, 2015 Accepted: March 28, 2015 Published: March 30, 2015
Citation: Ahmadi N, Nabavi V, Zughaib H, Patel N, Rathod A, et al. (2015) Aged Garlic Extract with Supplement is Associated with Beneficial Effect on Bone Mineral Density and Predicts Lack of Progression of Atherosclerosis: A Prospective Double Blinded Randomized Trial. Int J Cardiovasc Res 4:3. doi:10.4172/2324-8602.1000206

Abstract

 Aged Garlic Extract with Supplement is Associated with Beneficial Effect on Bone Mineral Density and Predicts Lack of Progression of Atherosclerosis: A Prospective Double Blinded Randomized Trial

Low levels of bone-mineral-density (BMD) are independently associated with the presence and severity of coronary-arterycalcium (CAC). This study evaluates the beneficial-effects of aged-garlic-extract therapy with-supplements (AGE-S) on levels of BMD, vascular-function, inflammation and CAC. Sixty subjects, randomized to four daily capsules of placebo vs. AGE-S inclusive of aged garlic extract (1000 mg) plus VitaminB12 (400 mcg or µg), folic-acid (1200 mcg or µg), Vitamin-B6 (50 mg) and L-arginine (400 mg) underwent CAC, thoracicBMD (mg/cc), lipid-profile, lipoprotein a (Lp-a), homocysteine and vascular-function measurement at baseline and 12- months. The postcuff-deflation temperature-rebound (TR), digital-thermal-monitoring index of vascular-function was assessed using a reactive-hyperemia-procedure. At 1-year, the mean increase in CAC and decrease in BMD was significantly lower in the AGE-S as compared to the placebo (p<0.05). After adjustment for risk-factors, the risk of CAC progression and reduced BMD was 65% and 68% less in AGE-S as compared to placebo (P<0.05). From baseline to 12 months, a significant correlation was noted between increase in CAC and decreases in BMD. Similarly, a significant correlation was noted between increase in TR and decrease in homocysteine as well as increase in Lp-a with lack of decrease in BMD. The maximum beneficial effect of AGE-S was noted with increase in TR, lack of decrease in BMD and lack of progression of CAC. In conclusion, this study demonstrates AGE-S is independently associated with favorable effect on A strong direct relation between increases in vascular-function, decrease in inflammation with lack of lowering in BMD levels as well as lack of CAC-progression in response to AGE-S was noted.

Keywords: Coronary artery calcium; Bone mineral density; Aged garlic extract; Vascular function; Inflammation; Computed tomography

Keywords

Coronary artery calcium; Bone mineral density; Aged garlic extract; Vascular function; Inflammation; Computed tomography

Introduction

The prevalence of osteoporosis and atherosclerosis are increasing worldwide as result of increase in life expectancy and often these two major health care problems coexist in both genders. Osteoporosis has been shown to be associated with cardiovascular disease [1], peripheral arterial disease [2], stroke and cardiovascular mortality [3].
Quantitative computerized tomography (CT) is a well-accepted method for non-invasive quantitative measurement of bone mineral density (BMD) levels [4]. Our recent study revealed an excellent agreement between thoracic vertebrae BMD levels and lumbar vertebrae BMD levels measured by CT (r2=0.97, p=0.0001) with high accuracy and reproducibility [4-6]. The finding of these studies suggests the feasibility of simultaneous measurement of BMD levels and CAC from the same cardiac CT imaging which can reduce both costs and radiation dose [5].
Previous studies revealed that aged garlic extract plus supplement (AGE-S) is associated with lack of progression of coronary atherosclerosis, increase in nitric oxide, improvement of vascular function and favorable effects on oxidative biomarkers [7]. However, the potential impact of AGE-S on osteoporosis has not been studied. In this randomized study, we evaluated the effect of AGE-S on BMD levels, inflammatory markers, vascular function and coronary artery calcium (CAC).

Subjects and Methods

This study is inclusive of 65 asymptomatic participants -aged 40-79 years- with Framingham risk scores (FRS) of 10-20% [8] and coronary artery calcium (CAC) >30 Agatston score, who were on chronic statin therapy and were free of clinical coronary artery disease (CAD). Participants were randomized to four capsules of either placebo or AGE-S. AGE-S consists of AGE (250 mg), vitamin B6 (12.5 mg), B12 (100 μg), folate (300 μg) and L-arginine (100 mg) (Kyolic 108, Wakunaga Nutritional Supplement, CA, USA).
All subjects received cardio-protective lifestyle education and their digital thermal monitoring (DTM) of vascular function, homocysteine lipoprotein a (Lp-a), lipid profile, BMD levels and CAC were measured at baseline and 12-month follows up, 60 subjects completed the study. Demographics, blood pressure and routine blood work were assessed using standard techniques. The study protocol (NCT00860847) and consent form were approved by the IRB Committee Board of the Los Angeles Biomedical Research Institute at Harbor UCLA Medical Center, Torrance, CA.
CAC scanning was performed with an E-Speed electron beam scanner (EBCT) (GE-Imatron, South San Francisco, Calif., USA). The coronary arteries were imaged with 30-40 contiguous 3 mm slices during mid-diastole using ECG-triggering during a 15 second breath hold. CAC was considered present in a coronary artery when a density of >130 Hounsfield units (HU) was detected in >3contiguous pixels (>1 mm2) overlying that coronary artery and was quantified using the previously described Agatston scoring method. CAC progression was defined as annual CAC progression >15% [9].
BMD levels of 4 consecutive thoracic (T) vertebrae (T-7 to T-10) were measured using Z axis (sagittal views). An automated region of interest (ROI) with diameter of 6 mm was located at the center of the trabecular region in each vertebral body, and average thoracic BMD levels in HU were measured (Figure 1). After calibration of measured BMD levels in HU with phantoms - using the N-Vivo bone densitometry application (Image Analysis, Kentucky) - BMD levels in mg/cm3 were calculated. The intra- and inter-observer variability of repeated thoracic BMD levels measurements were 2.5% and 2.8%, respectively [10].
Figure 1: Thoracic bone mineral density measured from nonenhancedcoronary artery calcium CT scans. The coronary calciumscore (green circle) was measured, and the BMD (red square) wasmeasured in four consecutive thoracic trabecular vertebrae (T-7 toT-10) below the slice level of left main coronary (green arrow). The10-mm region of interest (ROI) inside trabecular bone was used tomeasure BMD levels. The calibration phantom (yellow arrow) wasused to calibrate the CT scanner to measured density in mg/cm3.
Digital thermal mentoring of vascular function was measured in the morning in a quiet, dimmed room at a controlled ambient temperature of 23.5°C to 25.0°C after an overnight fast of 10 hours. The measurements were obtained with the subjects in the supine position and after 30 minutes of rest. Subjects’ blood pressure in the control arm was recorded in the sitting position 5 minutes before the DTM test. DTM of vascular function was obtained during 5 minutes of stabilization, 5 minutes of cuff inflation to 50 mm Hg greater than systolic blood pressure, and 5 minutes of deflation using an automated, operator-independent protocol (VENDYS, Endothelix, and Houston, Texas). Thermal changes during the 5-minute arm cuff-induced reactive hyperemia test were monitored continuously in the fingertip using VENDYS software. Vascular function was measured based on the amount of temperature rebound under area under the curve (TMP-AUC) in the occluded fingertip during the reactive hyperemia procedure.
Statistical Analysis
All statistical analyses were performed using SAS 9.2 (www.sas.com, Cary, NC) and STATA 12.1 (www.stata.com, College Station, Texas). All continuous data are presented as a mean value ± SD, and all categorical data are reported as a percentage or absolute number. Student's t tests and Chi-square tests were used to assess differences between groups. Logistic regression analyses were employed to assess the change in BMD level, homocysteine, Lp-a, lipid profile, vascular function and CAC in response to AGE-S. These analyses were adjusted for demographics, age, gender, conventional cardiovascular risk factors, body mass index and statin therapy.

Results

At baseline, there were no significant differences in cardiovascular risk factors, age, gender, CAC, homocysteine, Lp-a, lipid profile, TR and levels of BMD between the groups (Table 1 and 2)(P > 0.05). After 1 year of the study, the mean decrease in levels of BMD was significantly lower in the AGE-S as compared to the placebo group (p<0.05). Similarly, significant increases in TR, high density lipoprotein (HDL-C), Lp-a, decreases in total cholesterol, low density lipoprotein (LDL-C), triglyceride and homocysteine as well as lack of increase in CAC was noted in the AGE-S as compared to the placebo group (Table 2) (p<0.05). After adjustment of conventional risk factors using logistic regression, the risk of CAC progression and reduced BMD levels was 65% and 61% less in AGE-S as compared to placebo groups (P<0.05). AGE-S was associated with 13% reduction in LDL-C, 9% increase in HDL-C, 8% decrease in total cholesterol, 37% decrease in homocysteine and 78% increase in Lp-a as compared to placebo groups (p<0.05) (Table 3) The likelihood of combined increased in TR and BMD levels was 3.24 folds higher in AGE-S as compared to placebo. Similarly, likelihood of combined increased BMD levels and decrease in homocysteine was 2.39 folds higher with AGE-S. The likelihood of lack of progression in CAC and lack of regression with AGE-S was 9.1 folds higher as compared with Placebo. After adjustment for risk factors, the likelihood of simultaneous increased TR, decreased homocysteine, and lack of reduction in BMD levels and CAC progression was 14.99 folds higher with AGE-S as compared with placebo (Table 4) (p<0.05).
Table 1: Baseline demographic characteristics of study subjects..
Table 2: Baseline, 1-year follow-up and annual change in different levels of bone mineral density, temperature rebound, homocysteine and coronary artery calcium.
Table 3: The effect of AGE-S on levels of bone mineral density, temperature rebound, homocysteine and coronary atherosclerosis (Logistic Regression Analysis: Adjusted for age, gender, diabetes mellitus, Hypertension, hypercholesterolemia, Family History of CHD, Smoking status, Statin therapy and BMI, †Relative risk of CAC progression (increase in CAC ≥ 15%/year), ΔRelative risk of each standard deviation increase in BMD levels, temperature rebound and homocysteine..
Table 4: The linkage between change in levels of bone mineral density, temperature rebound, homocysteine and coronary atherosclerosis in response to AGE-S (Logistic Regression Analysis: Adjusted for age, gender, diabetes mellitus, Hypertension, hypercholesterolemia, Family History of CHD, Smoking status, Statin therapy and BMI, †Relative risk of CAC non-progression vs. CAC progression (increase in CAC ≥ 15%/year), ΔRelative risk of median increase in BMD levels, temperature rebound and decrease in homocysteine).
From baseline to 12 months, there was a significant correlation between decreases in BMD and increases in CAC and (r2=0.52, p=0.0001) (Figure 2). A significant correlation was noted between increases in levels of BMD and increases in TR (r2=0.83, p=0.001) and Lp-a (r2=0.52, p=0.001) as well as decreases in homocysteine (r2=0.65, p=0.0001). The significant interaction between increased in TR and decreased in homocysteine, as well as increase in Lp-a, with lack of decrease in BMD in response to AGE-S was noted (r2=0.89, p=0.01). Similarly, the significant interaction between increased in TR and Lp-a, decreased in homocysteine and increased in BMD levels with lack of CAC progression (r2=0.95, p=0.01). Maximum beneficial effect of AGE-S was noted with increase in TR, lack of decrease in BMD and CAC non-progression (Figure 3).
Figure 2: Aged garlic extract plus supplement is associated with lack of reduction in bone mineral density levels and progression of coronary artery calcium.
Figure 3: Maximum aged garlic extract plus supplement is noted with combined increase in temperature rebound, decrease in homocysteine and lack of progression of osteoporosis and coronary atherosclerosis.

Discussion

The current study demonstrates that: 1) there is a strong direct relationship between the lowering in BMD levels, and CAC progression; 2) AGE-S is independently associated with lack of lowering in BMD levels, lowering LDL-C and increase in HDL-C, decrease in inflammatory biomarkers and CAC non-progression, 3) A strong relation between increases in vascular-function, decrease in inflammation and lack of lowering in BMD levels in response to AGES was noted, and 4) AGE-S associated with decrease in homocysteine, increase in lipoprotein a and temperature rebound which predicted lack of lowering in BMD levels and CAC non-progression.
Osteoporosis and atherosclerosis are prevalent and are a major public health concern. Both are inflammation diseases which the inflammatory processes and vascular endothelial function plays an important role in their pathogenesis and progression [11-13]. Inflammatory cytokines such as IL-1ß and IL-6, in addition to their role in atherosclerosis, are involved in bone turnover and IL-1ß is a powerful stimulant of bone resorption; acting directly on osteoclasts and osteoclast precursors [12,13].
Increased baseline levels of Lp-a, a major carrier of oxidized phospholipids (OxPL), predict cardiovascular disease and clinical outcomes [14]. Increase in homocysteine and decrease in nitric oxide in response to inflammatory processes link osteoporosis and atherosclerosis. Hyperhomocysteinemia is associated with decrease in nitric oxide, endothelial cell ischemia, vascular endothelial dysfunction and plaque vulnerability [15,16].
Previous studies showed that endothelial nitric oxide synthase (eNOS) deficiency caused a significant reduction in bone mass in mice [17], whereas activation of nitric oxide and Akt, stimulated bone morphogenetic protein-2 (BMP-2) transcription and osteoblast differentiation [18,19].
Aged garlic therapy is associated increase bioavailability of eNOS, decrease in blood pressure platelet aggregation and adhesion, oxidized LDL-C, increase in HDL-C, associated with lack of progression of atherosclerosis [20,21]. B Vitamins and L-arginine also increase NO bioavailability, and favorable effects on oxidative stress. AGE-S has been shown to augment the beneficial effects of AGE [22].
Vascular endothelial function in addition to its known atheroprotective effects plays a role in osteoblast function and bone turnover [13] as well as regulation of bone remodeling by stimulating osteoblast differentiation, survival as well as the progression of osteoporosis [23]. Furthermore, the impaired vascular endothelial function is associated with the presence and severity of subclinical coronary atherosclerosis measured as CAC as well as increased risk of subsequent atherosclerotic cardiovascular disease events [24]. This study confirms previous studies and provided evidence that of linkage of improve in vascular endothelial function and homocysteine with lack of lowering in BMD levels in response to AGE-S.
Recent studies report pleiotropic effects of statin therapy including improvement of endothelial function, stabilization of atherosclerotic plaques, decrease in oxidative stress and inflammation, inhibition of the thrombogenic response and beneficial effects on immune system, CNS, and bone. Statin therapy activates AMP-activated protein kinase (AMPK), ROK, phosphorylate protein kinase B (Akt); resulting in activation of eNOS and increase in nitric oxide production and BMP-2 [25,26]. By inhibition of HMG-CoA reductase and ROK in osteoblasts, statins enhances BMP-2 and eNOS expression; stimulating bone formation, acting as an anabolic for bone and promoting the mineralization of osteoblasts [25]. As a result, statin therapy simultaneously increases the bone and prevents atherosclerosis [27]. A similar anti-inflammatory and anti-oxidative effect through nitric oxide pathway was noted in AGE-S [28].
The present study has extended previous observations of the role of AGE-S beyond lowering LDL-C and improving HDL-C; providing evidence, increase in Lp-a in response to AGE-S, similar to statin therapy, is associated with concomitant removal of OxPL from the vessel wall and the stabilization of atherosclerotic plaque, correlate strongly with increases in vascular function, regression in the burden of atherosclerosis [7].
This study reveals direct association between changes in CAC and BMD levels in response to AGE-S. Furthermore, increases in vascular function and decrease in homocysteine as well as increase in Lp-a in response to AGE-S is associated with lack of lowering in BMD levels and lack of CAC progression. These salutary effects have been associated with both concomitant stabilization of atherosclerosis [29], and positive changes in BMD levels, which stimulates osteoblast proliferation, differentiation, mineralization and survival.

Clinical Implication

Lower BMD levels are independently associated with increased risk of atherosclerosis, [30] stroke [31] and cardiovascular death [32]. Barengolts et al. [33] in their matched case control study on 45 postmenopausal women with and without low BMD levels reported that the mean CAC was significantly higher in those with lower levels of BMD as compared to matched control group independent of age and body mass index. The current study highlights the important role of AGE-S in simultaneous cardioprotection and prevention of osteoporosis by improving endothelial function and its antiinflammatory effects.
This study has several limitations. The study sample size was small; however this study clearly demonstrates the beneficial effects of AGE-S on BMD levels, vascular endothelial function, inflammatory biomarkers and CAC progression. Further studies are needed to assess the long-term effect of AGE-S on BMD levels on major adverse cardiovascular events (MACE) as pathologic fractures.

Conclusion

AGE-S is associated with favorable effects on lipid profile, BMD levels, and predicted lack of CAC progression. AGE-S associated with decrease in homocysteine increase in temperature rebound and Lp-a which predicted lack of lowering in BMD levels and CAC nonprogression. This highlights the role of AGE-S in simultaneous cardioprotection and prevention of osteoporosis by improving endothelial function and its anti-inflammatory effects.

Financial Support

This study was supported by a grant from Wakanuga Inc. of America, the manufacturer of the garlic formulation used in this study.

Conflict of Interest

Dr. Budoff has received grant support from Wakanuga. Other coauthors have no disclosures.

Acknowledgments

All authors have made substantial contributions to the conception and drafting and have approved the final version of this manuscript.

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