http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; 24(4): 612–617 © 2014 Japan College of Rheumatology DOI: 10.3109/14397595.2013.851639
ORIGINAL ARTICLE
Subclinical reduced G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients: Relation to clinical characteristics, disease activity and metabolic syndrome Tamer Atef Gheita1, Sanaa Abdel Baky Kenawy2, Rehab Wafik El Sisi3, Heba Atef Gheita4, and Hossam Khalil5 Mod Rheumatol Downloaded from informahealthcare.com by Nyu Medical Center on 04/16/15 For personal use only.
1Department of Rheumatology, Faculty of Medicine, Cairo University, Cairo, Egypt, 2Department of Pharmacology, Faculty of Pharmacy, Cairo University,
Cairo, Egypt, 3Oral and Maxillofacial Surgery, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt, 4Department of Pharmacology, Atomic Energy Authorization, Cairo, Egypt, and 5Department of Ophthalmology, Faculty of Medicine, Beni-Sweif University, Beni-Sweif, Egypt Abstract
Keywords
Objective. Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway. The purpose of this study was to investigate the enzyme activity of G6PD in Rheumatoid Arthritis (RA) and Sjögren’s Syndrome (SS) patients not known to be deficient in this enzyme. It was also within the scope of the aim to find the relation of G6PD to the presence of metabolic syndrome (MetS) in these patients. Methods. Erythrocyte G6PD activity was evaluated in 40 RA patients, 30 SS patients and in 30 ageand sex-matched control. The clinical characteristics, disease activity score (DAS28), SS disease activity (SSDAI) and damage (SSDDI) indices and presence of MetS of the included patients were analyzed in relation to the enzyme level. Results. The G6PD activity in RA patients (7.72 3.57 U/g Hb) was significantly reduced compared to that in the SS patients (11.55 3.14 U/g Hb) and control (13.23 3.34 U/g Hb) especially those with MetS (4.61 1.84 U/g Hb) (p 0.001). There was a significant negative correlation of the G6PD activity with the disease duration and DAS28 (p 0.001). Conclusion. The results of this study, suggest that G6PD not only does not protect against MetS in RA, but may even be considered a risk factor for the development of this disorder. The identification of regulatory tools for G6PD activity may prove promising for treating the associated metabolic disorders and chronic inflammation in RA.
DAS28, G6PD activity, Metabolic syndrome, Rheumatoid arthritis, Sjögren’s Syndrome
Introduction Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which regulates numerous enzymatic reactions involved in various cellular actions; yet its physiological function is seldom investigated [1,2]. Being recognized as an antioxidant enzyme, overexpression of G6PD may protect vascular endothelial cells from increased oxidant stress and regulate the intracellular redox milieu [1]. The importance of G6PD in cell growth and development as well as disease progression has been shown [3,4]. Its deficiency is associated with an increased susceptibility to certain diseases including diabetes [5]. It is plausible that the reduced proliferative capacity of G6PD-deficient cells impairs the turnover of damaged parenchyma cells. This, together with increased oxidative damage, can undermine the normal physiological functions of various tissues [3]. With the advancements in the field of G6PD research, it has become certain that this enzyme is an indispensable component of antioxidant defense. Whether its deficiency plays a pathogenic role Correspondence to: Tamer A. Gheita, Department of Rheumatology, Faculty of Medicine, Cairo University, PO Box 12611, Orman, Gamaa Street, 12613 Al Jizah (Giza), Egypt. Tel: 002 01004567975. Fax: 0020225085372. E-mail: [email protected]
History Received 22 August 2013 Accepted 2 October 2013 Published online 5 November 2013
in diseases other than hemolytic disorders remains to be clearly defined [6]. This deficiency in G6PD activity and hence the disturbance in redox homeostasis can lead to dysregulation of cell growth and signaling, anomalous embryonic development, altered susceptibility to viral infection as well as increased susceptibility to degenerative diseases [4]. G6PD deficiency is the most common enzyme deficiency worldwide. According to world health organization (WHO) 7.5% of world population are carriers of G6PD deficiency and 2.9% are deficient [7,8]. This enzyme deficiency is very prevalent in Africa, America, the Mediterranean and East Asia [8] with moderate health risks and no significant effect on longevity. It is a housekeeping enzyme essential for basic cellular functions including protecting red cell proteins from oxidative damage [9]. G6PD seems to have a central role in protection from apoptosis induced by oxidating agents [10]. G6PD deficiency is the most common disease producing enzymopathy. It is usually asymptomatic and with oxidative stressors a hemolytic crisis may occur [11]. There is a growing recognition that G6PD enzyme has diverse nonenzymatic functions including cell mobilization, control of apoptosis, and interaction and regulation of oncogenes modulation [9,12]. Unexpectedly, evidence indicates that G6PD-deficient patients are protected against ischemic heart and cerebrovascular disease and retinal vein occlusion [13,14].
DOI 10.3109/14397595.2013.851639
G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients
The increasing epidemic of obesity and metabolic syndrome (MetS) is an important predictor of current health issues. It is not completely understood how these disorders could increase oxidative stress [15,16]. It was revealed that G6PD is highly expressed in adipocytes and provokes the dysregulation of lipid metabolism and the adipocytokine expression, resulting in insulin resistance with a major contribution to the pathogenesis of MetS [16,17]. It has been proposed that a high level of G6PD in adipocytes may mediate the onset of metabolic disorders in obesity by increasing the oxidative stress and inflammatory signals [16]. The aim of this study was to investigate the enzyme activity of G6PD in Rheumatoid Arthritis (RA) and Sjögren’s Syndrome (SS) patients not known to be deficient in this enzyme. It was also within the scope of the aim to find the relation of G6PD to the presence of metabolic syndrome (MetS) in these patients.
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Patients and methods Forty RA patients were included in the current study with definite RA diagnosed according to the 2010 ACR/EULAR classification criteria for RA [18] attending the Rheumatology outpatient clinics of Cairo University Hospitals. Thirty primary SS patients diagnosed according to the revised classification criteria for SS [19] were included. Full history taking, thorough clinical examination and laboratory investigations including the complete blood count, erythrocyte sedimentation rate, liver and kidney function tests, rheumatoid factor (RF) and anti-cyclic citrullinated peptide (Anti-CCP) were performed. Anti-Ro, anti-La, Schirmer test and oral examination were performed for SS patients. The level of G6PD activity was assessed. The presence of metabolic syndrome (MetS) was considered among the patients according to the Adult Treatment Panel (ATP III) criteria [20]. Disease activity score, in 28 joints (DAS28) [21] was calculated for RA patients. The SS disease activity index (SSDAI) and SS disease damage index (SSDDI) were assessed for SS patients [22]. Patients with genetic G6PD deficiency or other diseases that may affect the G6PD activity such as cancer or liver problems were excluded. None of the patients had shown clinical manifestations of favism or drug induced hemolysis before their enrollment in the study. Thirty age- and sex-matched healthy subjects were included in the study as control. All the laboratory investigations and G6PD were performed to the control subjects. The study has been approved by the local ethics committee and has been performed
in accordance with the 1964 Declaration of Helsinki. Informed consents were taken from the patients. Assessment of G6PD activity Using Beutler method [23], Enzyme activity was determined in a cell lysate using a plate-reader spectrophotometer (ThermoMax Microplate Reader, Molecular Devices) by measuring the rate of increase of absorbance at 340 nm from the conversion of NADP (Nicotinamide adenine dinucleotide phosphate) to NADPH by G6PD. Substrate concentrations used were 200 mol/L glucose-6phosphate and 100 mol/L NADP. The cells are prepared by centrifuging whole blood at 2 000g for 10 min at 4°C. Plasma and buffy coat are carefully removed, and red cells hemolyzed with 4 volumes of cold, distilled water. G6PD activity is evaluated in hemolysates and the values expressed as nanomols of NADPH formed per minute (IU) per gram Hb. Statistical analysis The data were collected, tabulated and analyzed by SPSS package version 15 (SPSS corporation, USA). Data was summarized as mean SD. Mann–Whitney tests was used for comparative analysis of 2 quantitative data. Nonparametric analysis of variance (Kruskal–Wallis) was used for comparison of more than two groups; Spearman’s correlation analysis was used for detection of the relation between 2 variables. A linear multiple regression analysis with G6PD levels as the dependent variable was performed. Results were considered significant at p 0.05.
Results The study included 40 RA patients (32 females and 8 males) with a mean age of 35.4 6.9 years. All patients were receiving MTX (Methotrexate) (5–17.5 mg/week), 32 were receiving hydroxycloroquine 400 mg/day and 8 low dose corticosteroids (5–10 mg/ day). 35 patients were receiving nonsteroidal anti-inflammatory drugs on irregular basis to help decrease the duration of morning stiffness. None of the patients was receiving acetyl salicylate. The RF was positive in 34 patients with Anti-CCP positive in 27. Thirty Sjögren syndrome (SS) patients (25 females and 5 males) with a mean age of 34.27 8.36 years were included in the present study. The SS patients had a mean anti-Ro level of (16.3 8.45 U/ml), anti-La (22.53 9.16 U/ml), Schrimer test
Table 1. Characteristics of the RA and SS patients as well as controls. Age (years) Disease duration (years) BMI ESR (mm/h) RBC ( 106/mm3) Hb (g/dl) WBC ( 103/mm3) Platelets ( 103/mm3) Creatinine (mg/dl) AST (U/L) ALT (U/L) G6PD (U/g Hb) MTX dose (mg/day) RAI Morning Stiffness (min) DAS28
613
RA patients (40)
SS patients (30)
Control (30)
P
35.4 6.9 6.06 5.5 26.98 6.01 87.5 24.46 4.05 0.39 11.16 1.46 7.17 2.87 385.43 132.88 0.79 0.14 29.98 13.15 31.8 15.58 7.72 3.57 9.81 2.36 17.23 11.66 37.6 36.91 3.98 0.98
34.27 8.36 3.63 2.16 25.45 2.51 28.57 12.81 4.58 0.4 12.83 0.86 6.4 2.2 327.87 102.29 0.54 0.13 27.23 7.11 25.87 12.18 11.55 3.14 – – – –
33.1 6.12 – 27.1 3.52 21.53 8.73 4.51 0.36 12.98 1.1 6.9 2.44 291.7 70.29 0.42 0.13 25.67 8.56 21.13 13.12 13.13 3.19 – – – –
0.42 – 0.39 0.001 0.001 0.001 0.26 0.003 0.001 0.23 0.008 0.001 – – – –
BMI, Body–mass index; DAS28, Disease activity index in 28 joints; RAI, Ritchie articular index; MTX, methotrexate; ESR, Erythrocyte sedimentation rate; RBC, Red blood cells; Hb, hemoglobin; WBC, White blood cells; AST, Aspartate transaminase; ALT, Alanine transaminase. p significantly different at p 0.05.
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Figure 1. The level of G6PD activity in RA patients with and without MetS, SS patients and control.
(8.9 2.73 mm), SSDAI (3.93 3.05) and SSDDI (3.47 2.39). RF was positive in 10 and anti-CCP positive in 3. Table 1 shows the characteristics of the RA and SS patients as well as control. The G6PD activity was significantly reduced in the RA patients compared to the SS patients and control (p 0.001); it was reduced in 5 (16.67%) control subjects while it was reduced in 10 (30%) of the SS patients and 35 (87.5%) of the RA patients. Rheumatoid patients with MetS (n 7) had a significantly lower G6PD activity (4.61 1.84 U/g Hb) compared to those without MetS (n 33) (8.38 3.51 U/g Hb) (p 0.001). Two SS patients had MetS with comparable level of G6PD to those without MetS. There was a tendency to a lower G6PD in SS patients; however, the difference from the control did not reach significance (p 0.08). Figure 1 demonstrates the G6PD activity in RA patients with and without MetS, SS patients and control. There was no significant difference in the studied parameters according to the gender of the patients or medications received. However, there was a tendency to lower G6PD enzyme activity in males (5.76 2.93 U/g Hb) compared to the female RA patients (8.21 3.59 U/g Hb). Metabolic syndrome was present in 4 males and 3 females with a lower G6PD activity in the males (3.66 1.67 U/g Hb) compared to the level in RA females with MetS (5.87 1.35 U/g Hb) (p 0.11). The correlation of the studied clinical and laboratory parameters with the G6PD activity in RA patients are shown in Table 2. The significant negative correlation of the G6PD with the disease duration, RAI and DAS28 are demonstrated in Figures 2–4. There was a significant correlation of the DAS28 with the MTX dose received by the patients (r 0.43, p 0.005). Regression analysis test showed that the independent variables (Age, disease duration, BMI, morning stiffness, RAI, DAS28 and presence of metabolic syndrome) would predict the decrease in G6PD level (p 0.001). In the SS patients, there was no significant correlation of the G6PD level and the studied parameters. There was a tendency to a negative
correlation of the G6PD level and the SSDDI (r -0.3, p 0.11). In the control, the G6PD was lower in the males (11.1 1.31 U/g Hb) compared to the females (12.97 3.9 U/g Hb) (p 0.06) and significantly correlated with the BMI (r 0.48, p 0.007).
Discussion Oxidative stress and impaired antioxidant systems have an important role in the etiology of RA, pathogenesis of joint tissue injury and chronic inflammation [24]. In the present study, the G6PD Table 2. Correlation of the G6PD activity with the clinical and laboratory characteristics of the RA patients. RA patients (N 40) Features r (p) Age (years) Disease duration (years) BMI ESR (mmHg/1st h) RBC ( 106/mm3) Hb (g/dl) WBC ( 103/mm3) Platelets ( 103/mm3) Creatinine (mg/dl) AST (U/L) ALT (U/L) MTX (mg/week) RAI Morning stiffness (min) DAS28
G6PD (U/g Hb) 0.22 (0.17) 0.5 (0.001) 0.09 (0.56) 0.27 (0.09) 0.04 (0.82) 0.23 (0.16) 0.07 (0.67) 0.16 (0.32) 0.11 (0.5) 0.3 (0.07) 0.06 (0.72) 0.12 (0.48) 0.5 (0.001) 0.19 (0.23) 0.53 (0.001)
BMI, Body–mass index; DAS28, Disease activity index in 28 joints; RAI, Ritchie articular index; MTX, methotrexate; ESR, Erythrocyte sedimentation rate; RBC, Red blood cells; Hb, hemoglobin; WBC, White blood cells; AST, Aspartate transaminase; ALT, Alanine transaminase. p significantly different at p 0.05.
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DOI 10.3109/14397595.2013.851639
G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients
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Figure 2. Correlation of the G6PD activity and the DAS28 in RA patients.
level was significantly reduced in the RA patients compared to the control. G6PD deficiency is a common enzymopathy affecting more than 400 million people worldwide [6]. Its gene promoter is highly polymorphic with almost 400 reported variants, conferring different levels of enzyme activity [25]. Its deficiency is one of the most common human genetic abnormalities [14] and is the commonest inborn error of metabolism [26]. Studies conducted on the G6PD activity in rheumatic diseases are scarce. G6PD-deficiency has been described in case reports on children with Kawasaki disease [27,28] and adult onset Stills disease [29]. In another study, G6PD activity was significantly higher in osteoporosis than in osteoarthritis patients [30]. In the present study the G6PD activity was reduced in 16.67% of the control subjects, 30% of the SS and 87.5% of the RA patients. The frequency of G6PD deficiency varies markedly among different racial groups. Population studies in the Middle East have shown remarkable variations in its prevalence rates; in Egypt (1%), Lebanon (2.13%), Syria (2.98%), Jordan (3.6%), Kuwait (5.51%), Iran (11.55%) [31], Oman (27%), United Arab Emirates (8.7%), Yemen (6.2%) [32,33] and has been as high as 70% for Kurdistanian Jews [31,34]. G6PD deficiency is found mainly in the tropical and subtropical regions of the world, with the
highest rates in the Middle East, Africa, Asia and the Mediterranean, being 12.1% in Italian control subjects [14] and 21.5% among a population of Chinese female heterozygotes [35]. This variation could be due to the difference in the exposure to environmental factors that provoke oxidative stress. It is recommended that a larger number of patients and control from different geographical areas are included and consider their phenotyping analysis that may be contributing to the health burden of the country. Such data will provide baseline information for further studies on some aspects of G6PD in Egypt. In the present study, the G6PD activity was lower in the males. The G6PD is X-linked, and so deficient variants are expressed more commonly in males than in females [36]. Deficiency of G6PD was reported in almost 1% of clinically normal blood donors mostly in males [8]. Clinical manifestations are generally milder in females and depend on the degree of mosaicism [6,37]. In the present study, there was a significant negative correlation between the erythrocyte G6PD activity and disease activity of RA. In a previous study it was found that markers of increased oxidative stress and impaired antioxidant capacity including G6PD were profound in RA and significantly reflected disease activity [38]. The role of oxidative stress in the pathogenesis of RA is
Figure 3. Correlation of the G6PD activity and the disease duration in RA patients.
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Figure 4. Correlation of the G6PD activity and the Ritchie articular index (RAI) in RA patients.
confirmed, and decreased G6PD activity can serve as surrogate marker for disease activity. In the present study, there was a significant correlation of the enzyme activity with the BMI in the control subjects which was not significant in the patients. The associated inflammation in RA patients may aid to explain the absence of correlation with the BMI. In adipocytes, oxidative stress and chronic inflammation are closely associated with metabolic disorders, including insulin resistance, obesity, cardiovascular disease and type-2 diabetes. The molecular mechanisms underlying these disorders have not been thoroughly elucidated. Overexpression of G6PD in adipocytes stimulates oxidative stress and inflammatory responses and promotes the expression of prooxidative enzymes which eventually leads to the dysregulation of adipocytokines and inflammatory signals. Secretory factors from G6PD-overexpressing adipocytes stimulate macrophages to express more proinflammatory cytokines leading to chronic inflammation of the adipose tissue [16]. In the present study, rheumatoid patients with MetS had a significantly lower G6PD activity compared to those without. G6PD deficiency and diabetes can aggravate each other and both could be etiologically associated [39]. The G6PD plays an important role in beta-cell function and survival. High-glucose-mediated decrease in G6PD activity may provide a mechanistic explanation for the gradual loss of beta cells in diabetics [40]. The identification of regulatory tools for G6PD activity may prove promising for treating metabolic disorders such as obesity-induced insulin resistance and chronic inflammation [16]. There was a nonsignificant decrease in G6PD activity in SS patients compared to the control which may denote a negligible role in this disease. The screening for G6PD as part of the routine workup of blood donors in areas with a high prevalence of its deficiency is supported [41]. The reduced G6PD activity would raise its provocative potential as a risk factor in the pathogenesis of RA and the accompanying development of MetS. This reduction would also reflect inherent disease activity. To our knowledge, this is the first study to investigate the G6PD activity in rheumatoid arthritis patients and confirm its relation to metabolic syndrome. However, the role of G6PD in SS is imperceptible. It is recommended to perform the study on a larger number of patients and include further investigation on the promoter polymorphisms of G6PD genes in RA and SS patients. The relatively small number of patients and lack of genetic studies are considered limitations to this work. It is
recommended that RA patients manage the components of MetS to avoid further reduction of the G6PD activity.
Conflict of interest None.
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ORIGINAL ARTICLE
Subclinical reduced G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients: Relation to clinical characteristics, disease activity and metabolic syndrome Tamer Atef Gheita1, Sanaa Abdel Baky Kenawy2, Rehab Wafik El Sisi3, Heba Atef Gheita4, and Hossam Khalil5 Mod Rheumatol Downloaded from informahealthcare.com by Nyu Medical Center on 04/16/15 For personal use only.
1Department of Rheumatology, Faculty of Medicine, Cairo University, Cairo, Egypt, 2Department of Pharmacology, Faculty of Pharmacy, Cairo University,
Cairo, Egypt, 3Oral and Maxillofacial Surgery, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt, 4Department of Pharmacology, Atomic Energy Authorization, Cairo, Egypt, and 5Department of Ophthalmology, Faculty of Medicine, Beni-Sweif University, Beni-Sweif, Egypt Abstract
Keywords
Objective. Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway. The purpose of this study was to investigate the enzyme activity of G6PD in Rheumatoid Arthritis (RA) and Sjögren’s Syndrome (SS) patients not known to be deficient in this enzyme. It was also within the scope of the aim to find the relation of G6PD to the presence of metabolic syndrome (MetS) in these patients. Methods. Erythrocyte G6PD activity was evaluated in 40 RA patients, 30 SS patients and in 30 ageand sex-matched control. The clinical characteristics, disease activity score (DAS28), SS disease activity (SSDAI) and damage (SSDDI) indices and presence of MetS of the included patients were analyzed in relation to the enzyme level. Results. The G6PD activity in RA patients (7.72 3.57 U/g Hb) was significantly reduced compared to that in the SS patients (11.55 3.14 U/g Hb) and control (13.23 3.34 U/g Hb) especially those with MetS (4.61 1.84 U/g Hb) (p 0.001). There was a significant negative correlation of the G6PD activity with the disease duration and DAS28 (p 0.001). Conclusion. The results of this study, suggest that G6PD not only does not protect against MetS in RA, but may even be considered a risk factor for the development of this disorder. The identification of regulatory tools for G6PD activity may prove promising for treating the associated metabolic disorders and chronic inflammation in RA.
DAS28, G6PD activity, Metabolic syndrome, Rheumatoid arthritis, Sjögren’s Syndrome
Introduction Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which regulates numerous enzymatic reactions involved in various cellular actions; yet its physiological function is seldom investigated [1,2]. Being recognized as an antioxidant enzyme, overexpression of G6PD may protect vascular endothelial cells from increased oxidant stress and regulate the intracellular redox milieu [1]. The importance of G6PD in cell growth and development as well as disease progression has been shown [3,4]. Its deficiency is associated with an increased susceptibility to certain diseases including diabetes [5]. It is plausible that the reduced proliferative capacity of G6PD-deficient cells impairs the turnover of damaged parenchyma cells. This, together with increased oxidative damage, can undermine the normal physiological functions of various tissues [3]. With the advancements in the field of G6PD research, it has become certain that this enzyme is an indispensable component of antioxidant defense. Whether its deficiency plays a pathogenic role Correspondence to: Tamer A. Gheita, Department of Rheumatology, Faculty of Medicine, Cairo University, PO Box 12611, Orman, Gamaa Street, 12613 Al Jizah (Giza), Egypt. Tel: 002 01004567975. Fax: 0020225085372. E-mail: [email protected]
History Received 22 August 2013 Accepted 2 October 2013 Published online 5 November 2013
in diseases other than hemolytic disorders remains to be clearly defined [6]. This deficiency in G6PD activity and hence the disturbance in redox homeostasis can lead to dysregulation of cell growth and signaling, anomalous embryonic development, altered susceptibility to viral infection as well as increased susceptibility to degenerative diseases [4]. G6PD deficiency is the most common enzyme deficiency worldwide. According to world health organization (WHO) 7.5% of world population are carriers of G6PD deficiency and 2.9% are deficient [7,8]. This enzyme deficiency is very prevalent in Africa, America, the Mediterranean and East Asia [8] with moderate health risks and no significant effect on longevity. It is a housekeeping enzyme essential for basic cellular functions including protecting red cell proteins from oxidative damage [9]. G6PD seems to have a central role in protection from apoptosis induced by oxidating agents [10]. G6PD deficiency is the most common disease producing enzymopathy. It is usually asymptomatic and with oxidative stressors a hemolytic crisis may occur [11]. There is a growing recognition that G6PD enzyme has diverse nonenzymatic functions including cell mobilization, control of apoptosis, and interaction and regulation of oncogenes modulation [9,12]. Unexpectedly, evidence indicates that G6PD-deficient patients are protected against ischemic heart and cerebrovascular disease and retinal vein occlusion [13,14].
DOI 10.3109/14397595.2013.851639
G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients
The increasing epidemic of obesity and metabolic syndrome (MetS) is an important predictor of current health issues. It is not completely understood how these disorders could increase oxidative stress [15,16]. It was revealed that G6PD is highly expressed in adipocytes and provokes the dysregulation of lipid metabolism and the adipocytokine expression, resulting in insulin resistance with a major contribution to the pathogenesis of MetS [16,17]. It has been proposed that a high level of G6PD in adipocytes may mediate the onset of metabolic disorders in obesity by increasing the oxidative stress and inflammatory signals [16]. The aim of this study was to investigate the enzyme activity of G6PD in Rheumatoid Arthritis (RA) and Sjögren’s Syndrome (SS) patients not known to be deficient in this enzyme. It was also within the scope of the aim to find the relation of G6PD to the presence of metabolic syndrome (MetS) in these patients.
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Patients and methods Forty RA patients were included in the current study with definite RA diagnosed according to the 2010 ACR/EULAR classification criteria for RA [18] attending the Rheumatology outpatient clinics of Cairo University Hospitals. Thirty primary SS patients diagnosed according to the revised classification criteria for SS [19] were included. Full history taking, thorough clinical examination and laboratory investigations including the complete blood count, erythrocyte sedimentation rate, liver and kidney function tests, rheumatoid factor (RF) and anti-cyclic citrullinated peptide (Anti-CCP) were performed. Anti-Ro, anti-La, Schirmer test and oral examination were performed for SS patients. The level of G6PD activity was assessed. The presence of metabolic syndrome (MetS) was considered among the patients according to the Adult Treatment Panel (ATP III) criteria [20]. Disease activity score, in 28 joints (DAS28) [21] was calculated for RA patients. The SS disease activity index (SSDAI) and SS disease damage index (SSDDI) were assessed for SS patients [22]. Patients with genetic G6PD deficiency or other diseases that may affect the G6PD activity such as cancer or liver problems were excluded. None of the patients had shown clinical manifestations of favism or drug induced hemolysis before their enrollment in the study. Thirty age- and sex-matched healthy subjects were included in the study as control. All the laboratory investigations and G6PD were performed to the control subjects. The study has been approved by the local ethics committee and has been performed
in accordance with the 1964 Declaration of Helsinki. Informed consents were taken from the patients. Assessment of G6PD activity Using Beutler method [23], Enzyme activity was determined in a cell lysate using a plate-reader spectrophotometer (ThermoMax Microplate Reader, Molecular Devices) by measuring the rate of increase of absorbance at 340 nm from the conversion of NADP (Nicotinamide adenine dinucleotide phosphate) to NADPH by G6PD. Substrate concentrations used were 200 mol/L glucose-6phosphate and 100 mol/L NADP. The cells are prepared by centrifuging whole blood at 2 000g for 10 min at 4°C. Plasma and buffy coat are carefully removed, and red cells hemolyzed with 4 volumes of cold, distilled water. G6PD activity is evaluated in hemolysates and the values expressed as nanomols of NADPH formed per minute (IU) per gram Hb. Statistical analysis The data were collected, tabulated and analyzed by SPSS package version 15 (SPSS corporation, USA). Data was summarized as mean SD. Mann–Whitney tests was used for comparative analysis of 2 quantitative data. Nonparametric analysis of variance (Kruskal–Wallis) was used for comparison of more than two groups; Spearman’s correlation analysis was used for detection of the relation between 2 variables. A linear multiple regression analysis with G6PD levels as the dependent variable was performed. Results were considered significant at p 0.05.
Results The study included 40 RA patients (32 females and 8 males) with a mean age of 35.4 6.9 years. All patients were receiving MTX (Methotrexate) (5–17.5 mg/week), 32 were receiving hydroxycloroquine 400 mg/day and 8 low dose corticosteroids (5–10 mg/ day). 35 patients were receiving nonsteroidal anti-inflammatory drugs on irregular basis to help decrease the duration of morning stiffness. None of the patients was receiving acetyl salicylate. The RF was positive in 34 patients with Anti-CCP positive in 27. Thirty Sjögren syndrome (SS) patients (25 females and 5 males) with a mean age of 34.27 8.36 years were included in the present study. The SS patients had a mean anti-Ro level of (16.3 8.45 U/ml), anti-La (22.53 9.16 U/ml), Schrimer test
Table 1. Characteristics of the RA and SS patients as well as controls. Age (years) Disease duration (years) BMI ESR (mm/h) RBC ( 106/mm3) Hb (g/dl) WBC ( 103/mm3) Platelets ( 103/mm3) Creatinine (mg/dl) AST (U/L) ALT (U/L) G6PD (U/g Hb) MTX dose (mg/day) RAI Morning Stiffness (min) DAS28
613
RA patients (40)
SS patients (30)
Control (30)
P
35.4 6.9 6.06 5.5 26.98 6.01 87.5 24.46 4.05 0.39 11.16 1.46 7.17 2.87 385.43 132.88 0.79 0.14 29.98 13.15 31.8 15.58 7.72 3.57 9.81 2.36 17.23 11.66 37.6 36.91 3.98 0.98
34.27 8.36 3.63 2.16 25.45 2.51 28.57 12.81 4.58 0.4 12.83 0.86 6.4 2.2 327.87 102.29 0.54 0.13 27.23 7.11 25.87 12.18 11.55 3.14 – – – –
33.1 6.12 – 27.1 3.52 21.53 8.73 4.51 0.36 12.98 1.1 6.9 2.44 291.7 70.29 0.42 0.13 25.67 8.56 21.13 13.12 13.13 3.19 – – – –
0.42 – 0.39 0.001 0.001 0.001 0.26 0.003 0.001 0.23 0.008 0.001 – – – –
BMI, Body–mass index; DAS28, Disease activity index in 28 joints; RAI, Ritchie articular index; MTX, methotrexate; ESR, Erythrocyte sedimentation rate; RBC, Red blood cells; Hb, hemoglobin; WBC, White blood cells; AST, Aspartate transaminase; ALT, Alanine transaminase. p significantly different at p 0.05.
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Figure 1. The level of G6PD activity in RA patients with and without MetS, SS patients and control.
(8.9 2.73 mm), SSDAI (3.93 3.05) and SSDDI (3.47 2.39). RF was positive in 10 and anti-CCP positive in 3. Table 1 shows the characteristics of the RA and SS patients as well as control. The G6PD activity was significantly reduced in the RA patients compared to the SS patients and control (p 0.001); it was reduced in 5 (16.67%) control subjects while it was reduced in 10 (30%) of the SS patients and 35 (87.5%) of the RA patients. Rheumatoid patients with MetS (n 7) had a significantly lower G6PD activity (4.61 1.84 U/g Hb) compared to those without MetS (n 33) (8.38 3.51 U/g Hb) (p 0.001). Two SS patients had MetS with comparable level of G6PD to those without MetS. There was a tendency to a lower G6PD in SS patients; however, the difference from the control did not reach significance (p 0.08). Figure 1 demonstrates the G6PD activity in RA patients with and without MetS, SS patients and control. There was no significant difference in the studied parameters according to the gender of the patients or medications received. However, there was a tendency to lower G6PD enzyme activity in males (5.76 2.93 U/g Hb) compared to the female RA patients (8.21 3.59 U/g Hb). Metabolic syndrome was present in 4 males and 3 females with a lower G6PD activity in the males (3.66 1.67 U/g Hb) compared to the level in RA females with MetS (5.87 1.35 U/g Hb) (p 0.11). The correlation of the studied clinical and laboratory parameters with the G6PD activity in RA patients are shown in Table 2. The significant negative correlation of the G6PD with the disease duration, RAI and DAS28 are demonstrated in Figures 2–4. There was a significant correlation of the DAS28 with the MTX dose received by the patients (r 0.43, p 0.005). Regression analysis test showed that the independent variables (Age, disease duration, BMI, morning stiffness, RAI, DAS28 and presence of metabolic syndrome) would predict the decrease in G6PD level (p 0.001). In the SS patients, there was no significant correlation of the G6PD level and the studied parameters. There was a tendency to a negative
correlation of the G6PD level and the SSDDI (r -0.3, p 0.11). In the control, the G6PD was lower in the males (11.1 1.31 U/g Hb) compared to the females (12.97 3.9 U/g Hb) (p 0.06) and significantly correlated with the BMI (r 0.48, p 0.007).
Discussion Oxidative stress and impaired antioxidant systems have an important role in the etiology of RA, pathogenesis of joint tissue injury and chronic inflammation [24]. In the present study, the G6PD Table 2. Correlation of the G6PD activity with the clinical and laboratory characteristics of the RA patients. RA patients (N 40) Features r (p) Age (years) Disease duration (years) BMI ESR (mmHg/1st h) RBC ( 106/mm3) Hb (g/dl) WBC ( 103/mm3) Platelets ( 103/mm3) Creatinine (mg/dl) AST (U/L) ALT (U/L) MTX (mg/week) RAI Morning stiffness (min) DAS28
G6PD (U/g Hb) 0.22 (0.17) 0.5 (0.001) 0.09 (0.56) 0.27 (0.09) 0.04 (0.82) 0.23 (0.16) 0.07 (0.67) 0.16 (0.32) 0.11 (0.5) 0.3 (0.07) 0.06 (0.72) 0.12 (0.48) 0.5 (0.001) 0.19 (0.23) 0.53 (0.001)
BMI, Body–mass index; DAS28, Disease activity index in 28 joints; RAI, Ritchie articular index; MTX, methotrexate; ESR, Erythrocyte sedimentation rate; RBC, Red blood cells; Hb, hemoglobin; WBC, White blood cells; AST, Aspartate transaminase; ALT, Alanine transaminase. p significantly different at p 0.05.
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Figure 2. Correlation of the G6PD activity and the DAS28 in RA patients.
level was significantly reduced in the RA patients compared to the control. G6PD deficiency is a common enzymopathy affecting more than 400 million people worldwide [6]. Its gene promoter is highly polymorphic with almost 400 reported variants, conferring different levels of enzyme activity [25]. Its deficiency is one of the most common human genetic abnormalities [14] and is the commonest inborn error of metabolism [26]. Studies conducted on the G6PD activity in rheumatic diseases are scarce. G6PD-deficiency has been described in case reports on children with Kawasaki disease [27,28] and adult onset Stills disease [29]. In another study, G6PD activity was significantly higher in osteoporosis than in osteoarthritis patients [30]. In the present study the G6PD activity was reduced in 16.67% of the control subjects, 30% of the SS and 87.5% of the RA patients. The frequency of G6PD deficiency varies markedly among different racial groups. Population studies in the Middle East have shown remarkable variations in its prevalence rates; in Egypt (1%), Lebanon (2.13%), Syria (2.98%), Jordan (3.6%), Kuwait (5.51%), Iran (11.55%) [31], Oman (27%), United Arab Emirates (8.7%), Yemen (6.2%) [32,33] and has been as high as 70% for Kurdistanian Jews [31,34]. G6PD deficiency is found mainly in the tropical and subtropical regions of the world, with the
highest rates in the Middle East, Africa, Asia and the Mediterranean, being 12.1% in Italian control subjects [14] and 21.5% among a population of Chinese female heterozygotes [35]. This variation could be due to the difference in the exposure to environmental factors that provoke oxidative stress. It is recommended that a larger number of patients and control from different geographical areas are included and consider their phenotyping analysis that may be contributing to the health burden of the country. Such data will provide baseline information for further studies on some aspects of G6PD in Egypt. In the present study, the G6PD activity was lower in the males. The G6PD is X-linked, and so deficient variants are expressed more commonly in males than in females [36]. Deficiency of G6PD was reported in almost 1% of clinically normal blood donors mostly in males [8]. Clinical manifestations are generally milder in females and depend on the degree of mosaicism [6,37]. In the present study, there was a significant negative correlation between the erythrocyte G6PD activity and disease activity of RA. In a previous study it was found that markers of increased oxidative stress and impaired antioxidant capacity including G6PD were profound in RA and significantly reflected disease activity [38]. The role of oxidative stress in the pathogenesis of RA is
Figure 3. Correlation of the G6PD activity and the disease duration in RA patients.
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Figure 4. Correlation of the G6PD activity and the Ritchie articular index (RAI) in RA patients.
confirmed, and decreased G6PD activity can serve as surrogate marker for disease activity. In the present study, there was a significant correlation of the enzyme activity with the BMI in the control subjects which was not significant in the patients. The associated inflammation in RA patients may aid to explain the absence of correlation with the BMI. In adipocytes, oxidative stress and chronic inflammation are closely associated with metabolic disorders, including insulin resistance, obesity, cardiovascular disease and type-2 diabetes. The molecular mechanisms underlying these disorders have not been thoroughly elucidated. Overexpression of G6PD in adipocytes stimulates oxidative stress and inflammatory responses and promotes the expression of prooxidative enzymes which eventually leads to the dysregulation of adipocytokines and inflammatory signals. Secretory factors from G6PD-overexpressing adipocytes stimulate macrophages to express more proinflammatory cytokines leading to chronic inflammation of the adipose tissue [16]. In the present study, rheumatoid patients with MetS had a significantly lower G6PD activity compared to those without. G6PD deficiency and diabetes can aggravate each other and both could be etiologically associated [39]. The G6PD plays an important role in beta-cell function and survival. High-glucose-mediated decrease in G6PD activity may provide a mechanistic explanation for the gradual loss of beta cells in diabetics [40]. The identification of regulatory tools for G6PD activity may prove promising for treating metabolic disorders such as obesity-induced insulin resistance and chronic inflammation [16]. There was a nonsignificant decrease in G6PD activity in SS patients compared to the control which may denote a negligible role in this disease. The screening for G6PD as part of the routine workup of blood donors in areas with a high prevalence of its deficiency is supported [41]. The reduced G6PD activity would raise its provocative potential as a risk factor in the pathogenesis of RA and the accompanying development of MetS. This reduction would also reflect inherent disease activity. To our knowledge, this is the first study to investigate the G6PD activity in rheumatoid arthritis patients and confirm its relation to metabolic syndrome. However, the role of G6PD in SS is imperceptible. It is recommended to perform the study on a larger number of patients and include further investigation on the promoter polymorphisms of G6PD genes in RA and SS patients. The relatively small number of patients and lack of genetic studies are considered limitations to this work. It is
recommended that RA patients manage the components of MetS to avoid further reduction of the G6PD activity.
Conflict of interest None.
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