Study subjects
We carried out a case-control study in a convenience sample of 94 subjects ≥ 60 years of age, 50 healthy, 20 men and 30 women (mean age 67.9 ± 6.5 years) and 44 with osteoporosis, 19 men and 25 women (mean age 69.7 ± 7.3 years). The subjects were community-dwelling Mestizo Mexican elderly living in Mexico City for 10 years or more. Informative brochures were distributed in the community specifying the objectives of the study and admission criteria.
All the women had intact uterus and the mean age in which their menopause began is the same between the two study groups. None of the subjects studied had been taking antioxidant supplementation (vitamins or minerals), hormone replacement therapy or antiosteoporotic medication for at least 6 months prior to the study, none had acute or chronic diseases, or was receiving prescription medications.
Both groups were well-nourished, Mini Nutritional Assessment (MNA) score was > 23.5, and caloric intake was between 2,000 and 2500 kcal per day, and the alimentation had the nutrients requirements (protein, fat, carbohydrate, vitamins and minerals) between Recommended Dietary Allowance (RDA) measured by 24-h dietary recalls and serum albumin > 35 g/L [
14,
15].
The subjects agreed to participate in the study after giving their informed consent. The Ethics Committee of the Universidad Nacional Autónoma de México (UNAM) Zaragoza Campus approved the research protocol for this study.
Measurements
The following anthropometric measurements were obtained: weight, height, body mass index (BMI). It was considered as overweight when BMI ≥ 27 [
16].
Weight was measured while the subject was wearing underwear and a clinical smock and in a fasted state (after evacuation). A Torino® scale (Tecno Lógica, Mexicana, México, TLM®) was used, calibrated before each weight measurement. Height was obtained with an aluminum cursor stadiometer graduated in millimeters. The subject was barefoot, back, and head in contact with the stadiometer in Frankfurt horizontal plane. BMI was calculated by dividing weight (in kilograms) by height (in square meters).
Bone mineral density (BMD) was obtained at the peripheral DXA in calcaneus using a portable Norland Apollo Densitometer
®. Osteoporosis was considered when subjects had a BMD of 2.5 standard deviations (SD) or more below the mean value for young adults (T score, ≥2.5) [
17].
Blood samples were collected after a 12-h fasting period by venopuncture and placed in vacutainer/siliconized test tubes containing a separating gel and no additives, and heparin as anticoagulant agent (Becton-Dickinson, Mexico City, Mexico). Blood samples containing heparin were analyzed using a hemoglobin test protocol (including hemoglobin, hematocrit, and leukocyte counts). The following serum quantifications were conducted: glucose; urea; creatinine; urate; albumin; cholesterol; triglycerides, and cholesterol high-density lipoproteins (HDL). These tests were used as screening measurements for diagnosis of clinically healthy subjects.
Hemoglobin levels were measured by cyanomethahemoglobin reaction procedure (cut-off points: in males, 12.17–17.26 g/dL, and in females, 11.48–16.25 g/dL). Hematocrit levels were assessed by microhematocrit procedure (cut-off points: males, 38–52%, females, 36–51%). Leukocyte count was done using Newbauer Chamber procedure (cut-off points: 3,500–10, 650/mm3).
All spectrophotometric tests were determined using an UV-visible spectrophotometer (Shimadzu, Columbia, MD, USA). Specifically, glucose levels were measured with glucose oxidase method (cut-off points: 63–120 mg/dL), urea levels were measured with Berthelot urease method (cut-off points: 9.5–47.0 mg/dL), creatinine levels by Jaffe method without deproteinization (cut-off points: males, 0.3–1.5 mg/dL, females, 0.3–1.3 mg/dL), and urate levels by uricase colorimetric method (cut-off points: males, 2.9–8.88 mg/dL, females, 2.5–8.7 mg/dL). Albumin levels were measured by bromocresol green technique (3.23–4.03 g/dL).
Cholesterol was analyzed using CHOD-PAP technique (cut-off points: 168–200 mg/dL) and triglycerides by GPO-Trinder technique (cut-off points: 89–190 mg/dL), whereas HDL were assessed employing the same technique for cholesterol after precipitation of low and very-low lipoproteins using a phosphotungstic acid/magnesium chloride solution (cut-off points: 42–77 mg/dL).
All reagents employed in biochemical tests were obtained from Randox Laboratories, Ltd. (Crumlin, Co, Antrim, UK); cut-off points for reference values for Mexican elderly persons were determined at the Gerontologic Clinical Research Laboratory of the Universidad Nacional Autónoma de México (UNAM) Zaragoza Campus in Mexico City [
18].
Blood samples containing heparin were subjected to plasma total antioxidant status (TAS), activity of red blood cell (RBC) superoxide dismutase (SOD) and glutathione peroxidase (GPx), and plasma TBARS assay. Artefactual formation of TBARS in the samples was prevented by adding 10 μL of 2-mM butylated hydroxytoluene (BHT) in ethanol at 95% immediately after centrifugation.
Total antioxidant status was carried out using ABTS+ (2,2'-azidodiethylbenzothiazolin sulphanate) radical formation kinetics (Randox Laboratories, Ltd). The presence of antioxidants in plasma suppressed the bluish-green staining of the ABTS+ cation, which was proportional to the antioxidant concentration level. Kinetics is measured at 600 nm.
The method employs xanthine and xanthine oxidase (XOD) to generate superoxide radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye. SOD activity was measured by degree of inhibition of the reaction (Randox Laboratories, Ltd). Kinetics was measured at 505 nm.
GPx catalyses oxidation of glutathione (GSH) by cumene hydroperoxide, in the presence of glutathione reductase (GR) and NADPH; oxidized glutathione (GSSG) is immediately converted into the reduced form with concomitant oxidation of NADPH to NADP+. Decrease in absorbance at 340 nm is measured (Randox Laboratories, Ltd).
We used thiobarbituric acid reacting substances (TBARS) assay. It was performed as described by Jentzsch et al. (1996) [
19]. In the TBARS assay, one molecule of malondialdehyde (MDA) reacts with two molecules of thiobarbituric acid (TBA) with production of a pink pigment with absorption at 535 nm. Amplification of peroxidation during the assay is prevented by the addition of the chain-breaking antioxidant BHT. Plasma (400 μL) or MDA standard (0.2–4 μmol/L) prepared by hydrolysis of 1,1,3,3-tetramethoxypropane (TMP) (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 (2 mmol/L) (Sigma Chemical Co.) in 12 × 75 mm tubes. Then we added 50 μL TBA reagent (0.11 mol/L in 0.1 mol/L NaOH) (Fluka Chem., Buchs, Switzerland) and mixed the contents; subsequently, the contents were incubated at 90°C for 45 min in a water bath. The tubes were put on ice to stop the reaction. TBARS were extracted once with 1000 μL n-butanol (Sigma Chemical Co.). The upper butanol phase was read at 535 nm and 572 nm to correct for baseline absorption in UV-visible spectrophotometer (Shimadzu, Columbia, MD, USA). MDA equivalents (TBARS) were calculated using the difference in absorption at two wavelengths and quantification was done with calibration curve.
The intra- and inter-assay variation coefficients were less 5% in all the determinations.
The within-run precision for the markers were as follows: LPO by TBARS assay 4.6%; erythrocyte SOD 3.8%; GPx and TAS with Randox Laboratories 6% and 4.3%, respectively. We calculated SOD/GPx ratio.
Alternative cut-off values for each parameter were defined on the basis of the 90
th percentile of young healthy subjects. A stress score [SS] ranging from 1 to 6 was used, representing the severity of biomarkers modifications; a score 1 was given to each value higher or lower than the cut-off. We categorize subjects as follows according to their scale in SS: with OxS if SS was > 3, and without OxS if SS was 0–3 [
20,
21].
Risk factors were measured: sex (female), age (≥ 70 years), oxidative stress (SS > 3), cigarette smoking (≥ 1 year in the present), alcohol intake (≥ 2 cups/day) and overweight (BMI ≥ 27).