Supplementation of ascorbic acid and alpha-tocopherol is useful to preventing bone loss linked to oxidative stress in elderly

Abstract

Objective

To determine the effect of ascorbic acid and alpha-tocopherol on oxidative stress and bone mineral density (BMD) in elderly people.

Design

A double-blind, controlled clinical assay was carried out in a sample of 90 elderly subjects divided into three age-paired random groups with 30 subjects in each group. Group Tx0 received placebo, group Tx1 received 500 mg of ascorbic acid and 400 IU of alpha-tocopherol, whereas group Tx2 received 1,000 mg of ascorbic acid and 400 IU of alpha-tocopherol, for a 12-month period.

Measurements

We measured thiobarbituric acid reactive substances (TBARS), total antioxidant status (TAS), superoxide dismutase (SOD), and glutation peroxidase (GPx); BMD was obtained on DXA of hip and spine before and after the 12-month treatment period with supplementation of vitamins C and E.

Results

We found a positive correlation between hip-BMD and SOD (r = 0.298, p <0.05) and GPx (r = 0.214, p <0.05). Also, a significantly lower decrease of LPO (p <0.05) was observed as linked with hip bone loss in the Tx2 group than in the Tx0 group.

Conclusions

Our findings suggest that that administration of 1,000 mg of ascorbic acid together with 400 IU of alpha-tocopherol could be useful in preventing or aiding in the treatment of age-related osteoporosis.

Introduction

All aerobic organisms, as a result of basic metabolic reactions, generate large quantities of superoxide radicals and hydrogen peroxide (H2O2), which can cause oxidative damage at the cellular level (1, 2). Thus, the organism possesses antioxidant homeostatic mechanisms to prevent attack of cellular components and the consequent oxidation of the latter, among which we find metal-trapping proteins and the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, as well as estrogen in the case of women, among others (3, 4). Additionally, as a complement to these endogenous mechanisms, vitamins such as A, C, and E contribute significantly to the antioxidant system (5, 6). When the antioxidant system is not efficient in counterarresting the formation of free radicals (FRs), highly reactive molecules cause oxidative damage to macromolecules such as DNA, lipids, proteins, and carbohydrates. As a consequence of the disequilibrium between antioxidants/oxidants, so-called oxidative stress (OxS) is promoted, which constitutes an etiological and physiopathological factor of many chronic-degenerative diseases, among which diabetes mellitus, high blood pressure, cataracts, atherosclerosis, liver diseases, rheumatoid arthritis, osteoporosis, several types of cancer are prominent (7, 8, 9, 10, 11, 12).

Bone tissue is formed and replaced in a constant manner by means of the complex interaction among minerals, hormones, and specialized cells. This process occurs in equilibrated fashion until ca. the age of 40 years; after this stage, the rate of bone resorption exceeds that of formation, causing a decrease in bone mass. This disequilibrium is what gives rise to osteoporosis, a generalized skeletal disorder characterized by bone mass reduction and deterioration in bone-mass quality, causing an increase in bone fragility and a greater risk of fractures (13, 14, 15).

It has been demonstrated that the main risk factors for osteoporosis comprise inadequate ingestion of calcium and phosphorus, vitamin D deficiency, estrogen deficiency in the climacteric, low weight, chronic consumption of certain drugs (corticosteroids), physical inactivity, smoking, and the consumption of alcoholic beverages (16, 17, 18, 19).

Recent studies have suggested that there is an association between bone mineral density (BMD) and oxidative stress (OxS) (20, 21). In this regard, it has been demonstrated that high H2O2 levels favor differentiation of osteoclastic cells from osteoclasts and inhibit differentiation of osteoblastic cells from osteoblasts, thus propitiating an accentuated diminution in OxS-related BMD (22, 23, 24). In addition, our research group evidenced a decrease in antioxidant activity in patients with osteoporosis (25). Arjmandi et al. (2002) demonstrated that administration of vitamin E exerts a beneficial effect on bone quality in old rats (26); however, the effect of antioxidant therapy on BMD has been scarcely approached in humans. Thus, the aim of the present study was to evaluate the effectiveness of the administration of alpha-tocopherol and ascorbic acid on OxS and BMD in older adults.

Methods

Design and participants

Screening and selection phase

Advertisements were distributed in the community specifying the objectives of the study, admission criteria and commitments. One hundred eighty elderly subjects agreed to participate in the study; however only 135 had the inclusion criteria. Candidates were requested to sign informed consent a letter specifying the reasons underlying their interest to participate as well as any circumstance that could facilitate or affect the compliance at the study.

Treatment phase

With previous informed consent, a double-blind controlled clinical assay was carried out in 135 elderly subjects healthy or with controlled chronic disease, which were randomly distributed into three groups (groups Tx0, Tx1, and Tx2) with 45 persons in each group. The research protocol was approved by the Ethics Committee of the Universidad Nacional Autónoma de México (UNAM) Zaragoza Campus.

Group Tx0 received a placebo with a pharmaceutical presentation similar to that of the treatment, whereas the elderly members of Group Tx1 were administered 500 mg of ascorbic acid and 400 IU of alpha-tocopherol. At the same time, Group Tx2 received 1,000 mg of ascorbic acid and 400 IU of alpha-tocopherol. Treatment was self-administered by oral via daily.

Follow-up phase

Included in the follow-up phase of the study, were supervision visits in dwelling-house two times to week by gerontological promoters (27, 28), besides, it was organized one meeting weekly between participants and members of the research team for strengthen the compliance of the treatment.

Fifteen persons each group did not finish the treatment (Lost to follow up), because, they underwent an acute disease and their physician prescribed several medications and stop the vitamins ingest. Finally, thirty subjects each group complied

Average age per group, body mass index (BMI), life styles, and menopausal age were similar. None of the participants had fracture history; the clinic diagnostics and medications of the participants are showed in the Table 1.

Table 1.

Clinical Characteristics of the Groups Study

	Tx 0 n = 30	Tx 1 n =30	Tx 2 n =30
Age	67.6 ± 7.3	68.2 ± 7.3	68.8 ± 8.5
Gender
Female (%)	19 (66)	26 (87)	20 (68)
Male (%)	11 (34)	4 (13)	10 (32)
Menopausal age	46.3 ± 5.2	48.2 ± 4.3	47.8 ± 6.5
Body Mass Index (BMI) (kg/m2)	27.2 ± 4.0	27.1 ± 3.7	27.7 ± 4.4
BMI < 22 (%)	2 (7)	3 (10)	3 (10)
BMI ≥22-27 (%)	18 (60)	14 (47)	15 (50)
BMI > 27 (%)	10(33)	13 (43)	12 (40)
Bone Mineral Density	0.84 ± 0.2	0.77 ± 0.1	0.79 ± 0.2
Lumbar Spine (g/cm2)
Bone Mineral Density	0.85 ± 0.1	0.83 ± 0.1	0.84 ± 0.1
HIP (g/cm2)
Smoke (≥7 cigarettes/week)
Positive (%)	2 (7)	0 (0)	3 (10)
Negative (%)	28 (93)	30 (100)	27(90)
Alcohol Ingestion
(≥ 2 cups/day)
Positive (%)	1(3)	2 (7)	1 (3)
Negative (%)	29 (97)	28 (93)	29 (97)
Physical Exercise (≥ 3 days/week)
Positive (%)	22 (73)	17(57)	16(53)
Negative (%)	8 (27)	13 (43)	14 (47)
Clinical diagnostic
Healthy	17 (57)	18 (60)	15 (50)
Diabetes mellitus type 2 (DM2)	3 (10)	3 (10)	2 (7)
Arterial hypertension (AH)	5 (16)	3 (10)	5 (17)
DM2 & AH	3 (10)	3 (10)	4 (13)
Others	2 (7)	3 (10)	4 (13)
Medication
None	12 (40)	9 (30)	12 (40)
Acid acetylsalicylic	6 (20)	9 (30)	3 (10)
Glibenclamide	6 (20)	6 (20)	6 (20)
Captopril	5 (16)	4 (13)	6 (20)
Enalapril	3 (10)	2 (7)	3 (10)
Glucosamine	3 (10)	2 (7)	2 (7)
Others	1 (3)	1 (3)	2 (7)

Blood sampling and preparation

Blood samples were collected by venopuncture after a 12-h fasting period and were placed in vacutainer and siliconized test tubes containing a separating gel without additives. Heparin and ethylenedinitrilotetraacetic acid (EDTA) were used as anticoagulant agents. Blood samples containing heparin were analyzed using complete hemoglobin test protocol (including hemoglobin, hematocrit, and leukocyte counts). Likewise TBARS, TAS, SOD, GPx activities.

The samples without anticoagulant agents were centrifuged at 3,500 rpm for 10min at 15°C-20°C with a centrifuge Eppendorf 5804 (Hamburg, Germany); the serum obtained was subjected to the following tests: glucose, blood urea nitrogen (BUN), creatinine, urate, albumin, cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C) concentrations. These tests were used as screening measurements for diagnosis of clinically healthy subjects.

Figure 1.

Diagram of the number of patients actively followed up at different times during the trial

From the samples with heparin, 600 µL of the whole blood were kept for red blood cell (SOD), 100 µL for red blood cell (GPx). Each sample later was centrifuged at 3,500 rpm during 10min at 15°C-20°C a centrifuge Eppendorf 5804 (Hamburg, Germany). From the obtained plasma 100 µL were taken for TAS and 1000 µL for TBARS.

Blood and biochemical analyses

Glucose, cholesterol, triglycerides, and HDL-C concentration levels were determined using an Autoanalyzer Vitalab Eclipse Merck (Dieren, The Netherlands). In particular, glucose levels were measured by the glucose oxidase method (cut-off points: 63–120 mg/dL), urea levels by the Berthelot urease method (cut-off points: 9.5–47 mg/dL), creatinine levels by the Jaffe method without deproteinization (cut-off points: males, 0.3–1.5 mg/dL; females, 0.3–1.3 mg/dL).

Cholesterol was analyzed using CHOD-PAP technique (cutoff points: 168–200 mg/dL), triglycerides by GPO-Trinder technique (cut-off points: 89-190 mg/dL), whereas HDL was assessed using the same technique used to analyze cholesterol after precipitation of low- and very low-density lipoproteins using a phosphotungstic acid/magnesium chloride solution (cut-off points: 42–77 mg/dL).

All reagents used in biochemical tests were obtained from Randox Laboratories, Ltd. (Crumlin, Co, Antrim, UK).

Plasma TBARS

The TBARS assay was prepared as described by Jentzsch et al. (1996) (29). In the TBARS assay, one molecule of malondialdehyde reacts with two molecules of thiobarbituric acid (TBA) and thereby produces a pink pigment with absorption peak at 535 nm. Amplification of peroxidation during the assay is prevented by the addition of the chain-breaking antioxidant, butyril hidroxy toluene (BHT).

Plasma (400µL) or malondialdehyde (MDA) standard (0.2-4 µmol/L) prepared by hydrolysis of 1,1,3,3-tetramethoxypropane (Sigma Chemical Co., St. Louis, MO, USA) was mixed with 400 µL orthophosphoric acid (0.2 mol/L) (Sigma Chemical Co.) and 50 µL BHT (2mmol/L) (Sigma Chemical Co.) in 12x72 mm tubes. A total of 50µL TBA reagent (0.11mol/L in 0.1 mol/L NaOH) (Fluka Chem., Buchs, Switzerland) then has been added and the contents were mixed. Subsequently, the contents were incubated at 90°C for 45 minutes in a water bath. The tubes then were kept on ice to prevent further reaction. TBARS were extracted once with 1000 µL n-butanol (Sigma Chemical Co.). The upper butanol phase was read at 535 nm and at 572 nm to correct for baseline absorption in UV-spectrophotometer Shimadzu UV-1601 (Kyoto, Japan). MDA equivalents (TBARS) were calculated by the difference in absorption at the two wavelengths and quantification was done with calibration curve.

Plasma total antioxidant status (TAS)

Antioxidant quantification was done using 2, 2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS+) radical formation kinetics (Randox Laboratories, Ltd., Crumlin Co. UK). The antioxidants present in plasma suppressed the bluish-green staining of the ABTS+ cation, which was proportional to the antioxidant concentration level. The kinetics was measured at 600 nm with UV-spectrophotometer Shimadzu UV-1601 (Kyoto, Japan).

Red blood cell superoxide dismutase (SOD)

The method uses xantine and xantine oxidase (XOD) to generate superoxide radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolim chloride to form a red formazan dye. SOD activity was measured by degree of inhibition of the reaction (Randox Laboratories Ltd., Crumlin Co. UK). The kinetics was measured at 505 nm with UV-spectrophotometer Shimadzu UV-1601 (Kyoto, Japan).

Red blood cell glutathione peroxidase (GPx)

In the presence of glutathione reductase and NADPH, the oxidation of glutathione (GSH) by cumene hydroperoxide is catalyzed by GPx. Oxidized glutathione (GSSG) is immediately converted into the reduced form with a subsequent oxidation of NADPH to NADP+ (Randox Laboratories, Ltd., Crumlin Co. UK). Decrease in absorbance was measured at 340 nm with UV-spectrophotometer Shimadzu UV-1601 (Kyoto, Japan).

Bone mineral density

Subjects underwent dual-energy x-ray absorptiometry (DXA) in hip and lumbar spine, which was measured with a densitometer (Hologic model QDR-4000 S/N 55618) with a precision of 0.34%, taking for the osteoporosis-like criterion a value less than –2.5 standard deviations (SD) in the T scale of parameters obtained for young adults and evaluated by age, gender, and ethnic group. The technique for evaluating BMD was adjusted to that established by the manufacturer, and measurement was performed by a technician trained by a recognized private company.

Statistical analysis

Data were processed by standard statistical software SPSS 14.0 (SPSS Inc. Chicago, IL, USA). Descriptive statistics are analyzed by means ± standard error (SE). Results were analyzed using analysis of variance (ANOVA) and Dunnett test post hoc. A p value < 0.05 was considered significant.

Results

In Table 2, we can observe the glucose concentrations and lipid profile of the population under study before and after treatment, in which no statistically significant differences were found. With respect to the relation of BMD with OxS markers, a statistically significant positive correlation between HIP-BMD and SOD activity (r = 0.298, p <0.05) was observed, as well as of GPx (r = 0. 214, p <0.05) with BMD (Table 3).

Table 2.

Biochemical Characteristics after Antioxidant Supplement

	Healthy n = 20	Osteopenia n = 36	Osteoporosis n = 34
Glucose (mg/dL)
Baseline	97± 27.5	95± 32.0	90 ±18.4
12 months	102±22.0	113±45.8	106±38.2
Cholesterol (mg/dL)
Baseline	187± 23.1	199± 28.9	208 ±40.8
12 months	170±32.6	196±38.0	201±44.8
Triglycerides (mg/dL)
Baseline	177± 43.2	171 ±52.1	159± 51.5
12 months	150±80.0	176±102	165±73.3
HDL- Cholesterol (mg/dL)
Baseline	53.35±7.94	56.33±10.67	59.79±9.37
12 months	49.14±10.16	52.0±7.29	53.40±8.66

Table 3.

Correlation between Age, BMI, Oxidative Stress Markers and Hip BMD

		BMD	Age	LPO	CAT	SOD	GPx	SOD/GPx	BMI
r value	BMD	1.00	-0.383	0.100	0.189	0.411	0.446	-0.336	0.341
	Age		1.00	-0.166	-0.156	-0.289	-0.390	0.254	-0.618
	LPO			1.00	0.055	0.093	0.132	0.054	0.043
	TAS				1.00	-0.263	0.017	-0.252	0.401
	SOD					1.00	0.225	-0.033	0.284
	GPx						1.00	-0.853	0.302
	SOD/GPX							1.00	-0.337
	BMI								1.00
Sig.	BMD		0.027	0.313	0.178	0.019	0.011	0.046	0.044
(1-tailed)	Age			0.208	0.223	0.076	0.024	0.105	0.000
	LPO				0.394	0.326	0.260	0.396	0.417
	TAS					0.098	0.468	0.107	0.021
	SOD						0.135	0.436	0.080
	GPx							0.000	0.067
	SOD/GPx								0.046

LPO, Lipoperoxides; TAS, total antioxidant status; SOD, superoxide dismutase; GPx, glutation peroxidase

With regard to changes in OxS markers and BMD before and after treatment, we observed a statistically significant diminution in LPO concentration in addition to a statistically significant increase of DMO (Table 4).

Table 4.

Oxidative Stress Markers and Bone Mineral Density Differences after Antioxidant Supplement

Variable	Tx0 n = 30	Tx1 n= 30	Tx2 n= 30
LPO (mol/L)
Baseline	0.239±0.011	0.215±0.036	0.250±0.059
12 months	0.162±0.009	0.160±0.006	0.144±0.005
Differences	- 0.075 (31%)	- 0.052 (24%)	- 0.110 * (44%)
TAS (mmol/L)
Baseline	1.03±0.038	1.02±0.033	1.02±0.046
12 months	1.36±0.062	1.43±0.050	1.26±0.061
Differences	0.336	0.416	0.246
SOD (UI/L)
Baseline	175±1.16	175±1.23	176±1.24
12 months	176±2.66	175±2.27	175±2.11
Differences	0.571	- 1.292	- 1.714
GPx (UI/L)
Baseline	8188±558	8498±670	8025±487
12 months	8345±670	10416±976	10155±1086
Differences	- 104.7	1812.9	2314.6
Lumbar spine BMD (g/cm2)
Baseline	0.896±0.037	0.786±0.031	0.822±0.37
12 months	0.904±0.038	0.800±0.030	0.831±0.039
Differences	0.0083	0.0141	0.0095
Hip BMD (g/cm2)
Baseline	0.865±0.015	0.852±0.012	0.841±0.010
12 months	0.859±0.015	0.848±0.011	0.850±0.010
Differences	- 0.0056	- 0.0043	0.0087†

Lipoperoxides (LPO), total antioxidant status (TAS), superoxide dismutase (SOD) y glutation peroxidase (GPx); bone mineral density (BMD). Tx1 (placebo), Tx1 (500 mg of ascorbic acid and 400 IU of alpha-tocopherol), Tx2 (1,000 mg of ascorbic acid and 400 IU of alpha-tocopherol.). ANOVA * Tx1 versus Tx2 p = 0.007; †Tx0 versus Tx2 p = 0.047.

In Figure 2, the changes are shown of LPO levels before and after treatment, observing a significant proportional diminution (–44%) in group Tx2 in comparison with groups Tx0 and Tx1 (–31 and –24%, respectively). In terms of BMD, we found less bone loss at the hip level in group Tx2 (Figure 3).

Figure 2.

Proportional changes of LPO by treatment group. Tx0 –31%; Tx1 –24%; Tx2 –44%

Figure 3.

Differences of Hip BMD by treatment group. Tx0: -0.0056; Tx1: -0.0043 and Tx2 0.0087. ANOVA test, p< 0.05: Tx0 versus Tx2

Discussion

Osteoporosis is one of the most frequent chronic-degenerative disorders during old age and is the cause of up to 75% of bone fractures in individuals aged >50 years; therefore, it is consequently accompanied by a diminution in the quality of life (QOL) in elderly adults (30). Thus, the main objective of the treatment of osteoporosis should be the prevention of bone tissue loss, to avoid the risk of fractures, as well as the recuperation of bone mass and diminishing the pain and discomfort that can arise from fractures in patients with diagnosed osteoporosis (31).

Among the most frequently employed drugs in the prevention and treatment of osteoporosis, the following are highlighted: selective estrogen receptor modulators (SERMs); calcitonin-salmon; the bisphosphonates; calcium and vitamin D supplements, vitamin D-analogues, ipriflavone, strontium ranelate, and hormone replacement therapy (HRT) (14, 32, 33, 34).

On the other hand, it has been reported that persons with osteoporosis present the lowest levels of uric acid and of antioxidant enzymes (20), supporting the proposal that indicates antioxidant supplements with preventive and/or therapeutic aims for osteoporosis. In this regard, recent studies have demonstrated that the consumption of antioxidant vitamins can exert an influence on the quality of BMD on reducing the effects of OxS, which can be associated with bone mass loss; notwithstanding this, the scientific information available in humans is scarce and inconclusive (35, 36). Therefore, in the present study, we evaluated the influence of administration of alpha-tocopherol and ascorbic acid on OxS and BMD in older adults. On analyzing the biochemical markers of OxS per diagnosis, we observed that subjects with osteoporosis presented higher LPO values and a diminution of the activity of antioxidant enzymes SOD and GPx, although only a statistically significant difference was present in SOD activity. These findings support the hypothesis about link the bone tissue loss with deficit in the antioxidant system and OxS.

In this respect, our results coincide with those of other studies, in which a relationship has been observed between low antioxidant activity and an increase in bone resorption as a consequence of the increase of FR (37). Likewise, our research group found a directly proportional relationship between total serum antioxidant capacity and BMD, and demonstrated that OxS is a risk factor for osteoporosis in elderly adults because the SOD/GPx ratio increases (25, 38).

Another finding of the present study was the positive correlation between BMD and antioxidant enzyme activity. These results suggest that the efficiency of the enzymatic antioxidant system can prevent the decrease in age-relative BMD and with this, the risk of presenting osteoporosis. Our results coincide with that reported by Maggio et al. (2003), who in a study carried out in women >60 years of age, observed that antioxidant activity diminishes significantly in women with osteoporosis compared with healthy controls. In this respect, the authors a decrease in vitamins A, C, and E, uric acid, and the antioxidant enzymes SOD and GPx, as well as a positive correlation between BMD and GPx (23).

On the other hand, Sahni et al. (2008) found an effect protective of the vitamin C for bone tissue loss in elderly men, but not in elderly women (39), suggesting that this option therapeutic only can be useful for males.

On analyzing OxS markers by treatment, we observed that subjects exposed to Tx2 presented a significant effect on the concentration of LPO and BMD on the hip, which demonstrate that an intake of 1,000 mg of vitamin C in addition to 400 IU of vitamin E daily diminishes the concentration of LPO, favoring a slight increase in hip BMD. In this sense, it has been observed that the consumption of vitamins E and C can diminish bone resorption in non-smoking postmenopausal women (40). Similarly, Turan et al. (2003), in a study conducted in rats, found that the consumption of vitamins E and C combined with selenium can prevent the bone structural changes that present in osteoporosis (41). In this regard, it has been noted that the antioxidant-rich diet plays an important role in the prevention of osteoporosis, because if the alimentary intake of vitamins E and C is deficient, the risk of hip fracture increases significantly, while if the diet is rich in vitamins E, C, and D, as well as in carotenes and selenium, this risk decreases. However, it has also been reported that there is no relation between a diet rich in antioxidants and BMD; these inconsistencies justify continuing this line of research (42).

Finally, our findings support the proposal of the etiological and physiopathological association of OxS with osteoporosis and the possible beneficial effect of antioxidants as a coadjuvant in the prevention and treatment of osteoporosis.

Acknowledgements: This work was supported by grants from Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, and DGAPA, UNAM PAPIIT IN303009.

Financial disclosure: None of the authors had any financial interest or support for this paper.