Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait

Correspondence to: S O Olusi, Department of Pathology, Faculty of Medicine, Kuwait University, PO Box. 24923, Safat 13110, Kuwait. E-mail: olusoji@hsc.kuniv.edu.kw

OBJECTIVE: Obesity, defined as a body mass index (BMI) greater than 30 kg/m², is now recognised as a risk factor for diabetes mellitus, hyperlipidaemia, colon cancer, sudden death and other cardiovascular diseases. In this study, it is hypothesized that obesity is an independent risk factor for lipid peroxidation and decreased activities of cytoprotective enzymes in humans.

SUBJECTS: Fifty normal healthy subjects with healthy BMI (19-25 kg/m²) and 250 subjects with different grades of obesity (30-50 kg/m²) with no history of smoking or biochemical evidence of diabetes mellitus, hypertension, hyperlipidaemia, renal or liver disease or cancer.

MEASUREMENTS: To test this hypothesis, we assessed lipid peroxidation and cytoprotection by measuring the concentrations of plasma malondialdehyde (P-MDA) and the activities of erythrocyte copper zinc-superoxide dismutase (CuZn-SOD) and glutathione peroxidase (GPX).

RESULTS: The concentration of P-MDA was significantly lower (P<0.001) in subjects with healthy BMI (2.53±0.04 µmol/l) than in those with BMI above 40 kg/m² (4.75+0.05 µmol/l). Furthermore, there was a significantly positive association (r=0.342, P=0.013) between BMI and P-MDA. On the other hand, subjects with healthy BMI had significantly higher (P<0.001) erythrocyte CUZn-SOD (1464±23 units/g Hb) and GPX (98.4±3.3 units/g Hb) than those with BMI above 40 kg/m² (1005±26 units/g Hb) and (84.3±6.7 units/g Hb) respectively. Furthermore, erythrocyte CuZn-SOD and GPX activities were negatively associated with BMI (r=-0.566, P=0.005 and r=-0.436, P=0.018) respectively.

CONCLUSION: It is concluded from these results that obesity in the absence of smoking, diabetes mellitus, hyperlipidaemia, renal or liver disease causes lipid peroxidation and decreased activities of cytoprotective enzymes, and should therefore receive the same attention as obesity with complications.

International Journal of Obesity (2002) 26, 1159-1164. doi:10.1038/sj.ijo.0802066

obesity; plasma malondialdehyde; erythrocyte; copper; zinc; superoxide dismutase; glutathione peroxidase

Introduction

The degree of overweight can be expressed in several ways, but the most useful is the body mass index (BMI). This index is the body weight in kilograms divided by the square of the height in metres (weight (height)²). Healthy weight is defined as a BMI between 19 and 25 kg/m². Overweight is a BMI of 25-30 kg/m² and is associated with low risk. A BMI greater than 30 kg/m² is almost always associated with an increase in body fat and is synonymous with obesity, except in body builders and other athletes. Obesity is prevalent worldwide and it is associated with increased mortality, increased cardiovascular diseases, diabetes, colon cancers and gall bladder disease.^1,2,3,4 These relative risks range from 2 to 8 when the BMI is in the range 35-50 kg/m². This contrasts with the low risk to health of people with healthy body weight.⁵ Although the exact biochemical mechanisms responsible for the association between obesity and the above diseases have not been completely elucidated, it is known that increase in triglyceride stores is associated with a linear increase in the production of cholesterol which in turn is associated with increased cholesterol secretion in bile and an increased risk of gallstone formation and the development of gall bladder diseases.⁶ Similarly, increased levels of circulating triacylglycerol in obesity are associated with decreased concentrations of high-density lipoprotein,⁷ which may account for the increased risks for cardiovascular disease and heart attack in obese patients.

Some of the major characteristics associated with the hypertension of obesity in humans have also been studied extensively. They include the activation of the renin-angiotensin system,^8,9 high levels of circulating leptin,¹⁰ reduced growth hormone concentration,¹¹ and activation of the sympathetic nervous system.¹²

Although animal studies in rats have shown that obesity is associated with increased myocardial oxidative stress¹³ and increased lipid peroxidation,¹⁴ to the best of our knowledge there is no report in the literature of the effect of obesity per se on lipid peroxidation and erythrocyte cytoprotection in humans.

The objective of this study is to test the hypothesis that obesity per se causes increased plasma lipid peroxidation and decreased erythrocyte cytoprotection. Therefore, lipid peroxidation and erythrocyte cytoprotection were assessed by measuring the concentrations of plasma malondialdehyde (P-MDA) and the activities of erythrocyte copper zinc-superoxide dismutase (CuZn-SOD) and glutathione peroxidase (GPX) in 250 subjects with various grades of obesity but with no confounding factors, and comparing the values with those obtained for 50 age- and sex-matched subjects with healthy BMI.

Materials and methods

Subjects

The subjects used in this study were recruited from a house-to-house survey of the prevalence of obesity in the Kuwaiti population. Written informed consent was obtained from each subject before the study and the survey was approved by an Ethical Committee on Human Research. In the survey proper, a total of 7500 Kuwaitis aged 15 and above were visited in their homes by trained interviewers. Height was recorded using a measuring tape, with the individual standing straight without shoes next to the wall, with the heels, buttocks, shoulders and occiput touching the wall. The head was kept erect and the measuring tape was stretched slightly to measure the height to the nearest 0.1 cm. Weight was recorded on a measuring scale calibrated daily at the beginning of each working day. The individual was requested to wear light dress and the weight was recorded with the individual barefooted by taking two successive readings to the nearest 100 g, the mean of which was recorded. The BMI for each individual was calculated from the formula (weight in kg)/(height in metres)². From the data obtained, 50 subjects with healthy BMI (19-25 kg/m²) were chosen randomly and were age- and sex-matched with 50 subjects each with BMI in the range 30-34, 35-39, 45-49 and 50 kg/m², respectively. From the detailed history and routine biochemical investigations done on each subject during the survey, only obese subjects with no history of smoking or biochemical evidence of diabetes, hypertension, dyslipidaemia, renal or liver diseases were included in this study in order to find out whether obesity on its own is an independent risk factor for lipid peroxidation and depletion of erythrocyte cytoprotective enzymes.

Sample preparation

Venous blood was drawn from each subject, after identification as above, and after an overnight fast into ethylenediaminetetracetic acid (EDTA) tubes and centrifuged at 3000 rpm for 10 min at 4°C. Haemolytic, icteric or turbid samples were excluded. Plasma was separated for the assay of malondialdehyde. The buffy coat was removed and the remaining erythrocytes were removed from the bottom, washed three times in cold saline (9.0 g/l NaCl) and haemolysed by the addition of an equal volume of ice-cold demineralized ultrapure water (MilliQ plus reagent grade; Millipore) to yield a haemolysate. Five hundred microlitres (500 µl) of the aliquots were prepared for the assay of CuZN-SOD and GPX.

Assay of plasma¾malondialdehyde

Plasma MDA concentrations were assayed using reagents from Sobioda (Grenoble, France), which employ a fluorimetric technique that is based on the method developed by Yagi.¹⁵ Malondialdehyde produced by hydrolysis of lipid hydroperoxides when heated under acid conditions reacts with thiobarbituric acid (TBA) to form a red complex, which absorbs light maximally at 532 nm. The complex is usually measured after extraction into butanol (to improve sensitivity) and is quantified against malondialdehyde standards generated from tetramethoxypropane under the same reaction conditions. The generation of malondialdehyde from lipid hydroperoxides during the course of the test is dependent on the presence of trace amounts of iron in the reagent used.

Although decomposition of peroxidized lipids is the major source of malondialdehyde, some may originate from other sources. The major criticism of the TBA test has been that a number of biologically significant substances apart from malondialdehyde, including biliverdin, ribose, 2-amino-pyrimidines and sialic acid, may form a complex with TBA and can interfere in the reaction. The TBA test is more sensitive and specific when fluorimetric detection is used, although some interference can still occur. The method used in this study is based on linking two molecules of TBA with a molecule of MDA at 95°C in an acid environment. Fluorescence with an excitation of 532 nm and emission of 553 nm was monitored in an Aminco Bowman spectrofluorimeter (Aminco Bowman Instruments). The concentrations of P-MDA were read off a calibration curve prepared from ethanolic 1, 1, 3, 3-tetramethoxy propane. A quality control check was carried out using control sera. Within- and between-batch precisions were done using pooled plasma.

Measurement of haemoglobin concentration

In order to express the activities of CuZn-SOD and GPX per gram haemoglobin (Hb), Hb concentration was measured in the haemolysates. Hb concentration was measured with a Coulter MAXM (Beckman-Coulter Instruments, USA) based on the method recommended by the National Committee for Clinical Laboratory Standards.¹⁶ Haemoglobin reacts with Drabkin's reagent, which contains potassium ferricyanide, potassium cyanide and sodium bicarbonate. Most of the Hb present in the blood is converted to methaemoglobin by ferricyanide. The methaemoglobin reacts with cyanide to form cyanmethaemoglobin, the absorbance of which, at 540 nm, is proportional to the Hb content in the haemolysate. Haemoglobin standard was diluted, with Drabkin's reagent, in the range 0-18 g Hb/dl blood and the absorbance measured at 540 nm. An aliquot of Drabkin's reagent was used as blank.

Assay of erythrocyte CuZn-SOD

Measurement of CuZn-SOD activity was performed using reagents from Ransod (Randox Laboratories) and was based on the method developed by McCord and Fridovich¹⁷ coupling O₂ generators (xanthine and xanthine oxidase, XOD) with an O₂ detector (2-(4-iodopehyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride; INT). Absorbance was monitored using a Beckman DU 7500 spectrophotometer (Beckman Instruments) for 30 s (for the initial reading A1) after the addition of XOD (125µl) as the starting reagent and subsequently for 3 min for the final reading (A2). The final reaction volume was 1 ml. All rates for the calibrators and the diluted samples were converted into percentages of the rate for the sample diluent (uninhibited) and substracted from 100% to give a percentage of inhibition. The unit of activity of the assay was defined as the amount of CuZn-SOD that inhibited the rate of formazan dye formation by 50%. The activity of CuZn-SOD in units/l divided by Hb in grams/l gave the activity of CuZn-SOD in units/g Hb. The precision of the assay was checked with Ransod controls (Randox Laboratories).

Assay of erythrocyte GPX

Measurement of GPX was performed using the reagent from Ransel (Randox) and was based on the method of Paglia and Valentine.¹⁸ GPX catalyses the oxidation of reduced glutathione (GSH) by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione (GSSG) is immediately converted to the reduced form (GSH) with concomitant oxidation of NADPH to NADP+. The decrease in absorbance at 340 nm is measured.

Drabkin cyanide-ferricyanide solution, pH 7.0-7.4, was prepared by dissolving potassium ferricyanide (200 mg), potassium cyanide (50 mg), and potassium dihydrogen phosphate (140 mg) in distilled water, making the volume up to 1 litre, and mixing after the addition of 0.5 ml of 250 g/l Brij 35.

For the GPX assay, haemolysates (50 µl) were diluted with 1 ml of Ransel diluting agent (Ransel Reagent; Randox) and incubated for 5 min, followed by the addition of 1 ml of the Drabkin Reagent and mixing (total dilution of the haemolysates before assay, 1:82). The diluting agent reduces any GSSG present in the haemolysates to GSH because the cyanide in the Drabkin reagent also inhibits other peroxidases that may be present in human blood and prevents falsely high results.

One unit of GPX was defined as the amount of enzyme that catalysed the transformation of 1 µmol of NADPH per minute under the assay condition. The activity of GPX in units/l divided by Hb concentration in g/l gives the activity of GPX in units/g Hb. Precision of the assay was checked with Ransel control (Randox Laboratories).

Statistical analyses of data

Statistical analyses were performed using SPSS 9.0 for Window (SPSS Inc., Chicago, IL, USA). Differences in mean values between groups were evaluated by a one-way analysis of variance (ANOVA) and Student's t-test. Two-tailed P-values were used and statistical significance was considered at P<0.05. The association between variables was evaluated by linear regression. Data were expressed as mean±standard error of mean (s.e.m.).

Results

An evaluation of the within-run and between run precisions of the methods for P-MDA, erythrocyte CuZn-SOD and GPX showed that within-run and between-run precisions were less than 4.5 and 5.8%, respectively.

Demographic and biochemical profiles of the study subjects

Table 1 shows that the mean age, plasma glucose, total cholesterol, high density lipoprotein-cholesterol, low-density-lipoprotein cholesterol, triglyceride and creatinine were similar in all the groups, thus eliminating the confounding effects of age, diabetes, hyperlipidaemia and renal failure on lipid peroxidation.

Plasma malondialehyde and BMI

Table 2 shows the plasma concentrations of malondialdehyde in the subjects with healthy body weight and those with different grades of obsesity. The P-MDA concentration in subjects with healthy body weight (2.53±0.04 µmol/l) was significantly lower (P<0.001) than those with BMI greater than 40 kg/m² (4.75±0.15 µmol/l), suggesting that obesity on its own is an independent risk factor for plasma lipid peroxidation. Table 3 shows further that there is a positive association between BMI and plasma concentrations of malondialdehyde (r=0.342; P=0.013), suggesting also that the greater the body weight the greater is the risk of plasma lipid peroxidation.

Erythrocyte CuZn-SOD activity

Table 2 shows an inverse relationship between BMI and erythrocyte CuZn-SOD activity. The mean erythrocyte CuZn-SOD activity of subjects with healthy body weight (1464±23 units/g Hb) was significantly higher (P<0.001) than in subjects with BMI greater than 40 kg/m² (1035±26 units/g Hb). The table also shows that subjects with BMI above 50 kg/m² have the lowest activity of erythrocyte CuZn-SOD (853±30 units/g Hb). Table 3 shows a statistically significant negative association (r=-0.566; P<0.005) between erythrocyte CuZn-SOD and BMI, suggesting that obesity is associated with a decreased activity of the erythrocyte cytoprotective enzyme CuZn-SOD.

Erythrocyte GPX activity

Table 2 shows an inverse relationship between the erythrocyte cytoprotective enzyme GPX and BMI. The activity of this enzyme in individuals with healthy BMI (98.4±3.3 units/g Hb) was significantly lower (P<0.001) than the value in those with BMI greater than 40 kg/m² (84.3±6.7). Subjects with BMI greater than 50 kg/m² had the least activity of this enzyme (76.3±6.9 units/g Hb). Table 3 also shows that there is a significant negative association between BMI and erythrocyte GPX activity (r=0.436; P=0.018).

Discussion

Lipid peroxidation is a free radical-generating process which occurs on every membranous structure of the cell. Free radicals are known to be involved in a number of human pathologies including atherosclerosis,¹⁹ cancer²⁰ and hypertension.^21,22 In this study, we confirmed in humans the finding in experimental rat models^13,14 that severe obesity is associated with lipid peroxidation. What is probably of more significance in this study is that obesity alone, without the confounding factors like hypertension, diabetes, hyperlipidaemia and smoking, causes significant lipid peroxidation. There are at least three main ways by which obesity, acting independently, can produce lipid peroxidation. Obesity increases the mechanical and metabolic loads on the myocardium, thus increasing myocardial oxygen consumption. A negative consequence of the elevated myocardial oxygen consumption is the production of reactive oxygen species such as superoxide, hydroxy radical and hydrogen peroxides from the increased mitochondrial respiration.²³ If the production of these oxygen species exceeds the antioxidant capacity of the cell, oxidative stress resulting in lipid peroxidation may occur. The animal model of Vincent et al¹³ demonstrated this quite clearly and our results confirmed that the same process can occur in humans. The second mechanism by which obesity can independently cause increased lipid peroxidation is by progressive and cumulative cell injury resulting from pressure from the large body mass. Cell injury causes the release of cytokines especially tumour necrosis factor alpha, which generates reactive oxygen species from the tissues which in turn cause lipid peroxidation.²⁴ A third possible mechanism is through the diet. Nutritional obesity which is the predominant form in our study population implies the consumption of hyperlipidaemic diets which may be involved in oxygen metabolism. Double bonds in the fatty acid molecules are vulnerable to oxidation reactions and consequently may cause lipid peroxidation.

In a rat model of diet-induced obesity, Dobrian and his colleagues¹⁴ reported increases in the activities of erythrocyte CuZn-SOD and GPX after 10 weeks on the diet. They attributed the increases in these erythrocyte cytoprotective enzymes, which are antioxidants, to their stimulation by oxidative stress. Similarly, Vincent et al¹³ in their study of obese Fatty Zucker rats reported increased activities of erythrocyte CuZn-SOD and GPX. In this study, however, we found that in obese humans there was a decrease in the activities of these enzymes. Our results were in agreement, however, with those of Moor de Burgos,²⁵ who found decreased antioxidant levels in obese adults, and those of Kuno et al,²⁶ who found decreased levels in girls, Decsi et al,²⁷ who found decreased levels in boys, and Strauss,²⁸ who found decreased serum concentrations of -tocopherol and -carotine in obese compared with non-obese children. The discrepancy between our results and those of Dobrian et al and Vincent et al could be due to the duration of the obesity. It is likely that, in the early days of the development of obesity, antioxidant enzyme activity will be stimulated. However, once the obesity persists for a long time, as in humans, the sources of the antioxidant enzymes become depleted, leading to a low level of activity, as we found in our study. The consequence of the low activity of the cytoprotective enzymes in human obesity is progressive tissue damage, which may eventually lead to atherosclerosis, cancer and other diseases.

In conclusion, this study has demonstrated that obesity in humans, in the absence of other confounding factors such as smoking, hypertension, diabetes and hyperlipidaemia, is an independent risk factor for lipid peroxidation and depletion of cytoprotective enzymes.

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Table 1 Demographic and biochemical profiles of the study groups

Table 2 Concentrations of plasma malondialdehyde and activities of erythrocyte CuZn-SOD and GPX in normal and obsese subjects

Table 3 Correlations between plasma malondialdehyde, erythrocyte cytoprotective enzymes and body mass index in healthy subjects