IJETST- Vol.||04||Issue||01||Pages 4926-4933||January||ISSN 2348-9480

2016

International Journal of Emerging Trends in Science and Technology IC Value: 76.89 (Index Copernicus) Impact Factor: 2.838 DOI: https://dx.doi.org/10.18535/ijetst/v4i1.01

Gender Differences in Inflammatory Markers among Type 2 Diabetic Subjects Authors

Sowmya Rajashekar, Asna Urooj Department of Studies in Food science & Nutrition, University of Mysore, Mysore 570006, Karnataka, India Abstract In India currently around 40.9 million people are diabetic and is expected to rise to 69.9 million by 2025 unless preventive steps are taken. The so called “Asian Indian Phenotype” refers to certain unique clinical and biochemical abnormalities. Diabetes mellitus and cardiovascular diseases act as two sides of the same coin. According to Framingham study, diabetic women have an increased cardiovascular risk about 3.5 fold higher than non diabetic women. In view of the impact of sexual hormones on glucose homeostasis, the molecular pathways involved in insulin resistance suggest a gender specificity mechanism in the development of diabetic complications and also seems to progress at a faster rate in females compared to males and women benefit less from treatment than do men. Hence a case controlled study was done to mark the gender differences in inflammatory markers among newly detected and known type 2 diabetic subjects. The study included 33 women and 17 men in the NDM group and 30 women and 20 men in the DM5 group. The biomarkers namely homocysteine (µmol/l), C-reactive protein (mg/l), lipoprotein (a) (mg / dl) and vitamin B12 (pg/ml) were analyzed. It was observed that the Hcy and Lp(a) values were higher in males than the females but CRP and Vitamin B12 values were higher in women when compared to males. (24.54±12.59, 3.81±1.14, 15.67±14.22 and 280.69±77.03 and among the women it was 17.06 ±5.54, 5.84 ±3.47, 10.55 ±12.89 and 352.35± 238.02 respectively). Among the DM5 all the values of the inflammatory markers were higher in males than in females. (It was 24.59 ±5.13, 4.25 ±0.79, 32.70 ±14.54 and 343.43 ±178.39 and in women, 17.64 ±5.07, 3.68 ±0.51, 24.25 ± 9.40 and 338.40 ± 158.15 respectively) From these observations it can be suggested that there are marked differences among the gender in the values of the inflammatory markers. It can also be suggested that the process of inflammation begins on the onset of diabetes which can progress as the disease progresses. Thus periodic analysis is required to efficiently treat the inflammation process to avoid complications related to cardio vascular risk Keywords: Newly detected diabetes mellitus (NDM), known diabetes mellitus (DM5), homocysteine (Hcy), lipoprotein (a) (Lp(a)), C- reactive protein(CRP). Introduction The impact of diabetes on the incidence of CVD is high when compared to the non- diabetics. It is possible that precursors of CVD have a greater effect in those with diabetes than in non -diabetic individuals (1). According to WHO, the estimated diabetic patients in India are more than 20million, which is estimated to reach 55million by 2025. Studies have shown that there is an average annual increase in rate of heart disease from 7 per 1,000 to 68 per 1,000 (2). Despite earlier treatment, there Sowmya Rajashekar et al

is a continued incidence of CVD, in people with T2DM. In 1970’s the prevalence of diabetes was approximately 2% among urban population in India, but at present the prevalence is more than 12%. A recent study conducted in six different cities; support that prevalence rate is much higher in metropolitan cities – Hyderabad (16.6%), Chennai (13.5%), Bangalore (12.4%), Kolkata (11.7%), Delhi (11.6%), Mumbai (9.3%) (3).

www.ijetst.in

Page 4926

IJETST- Vol.||04||Issue||01||Pages 4926-4933||January||ISSN 2348-9480 The so called “Asian Indian Phenotype” refers to certain unique clinical and biochemical abnormalities in Indians which include increased insulin resistance, greater abdominal adiposity that is higher waist circumference despite lower body mass index, lower adiponectin and higher sensitivity C-reactive protein levels (4). Type 2 diabetes increases the risk of cardio vascular disease (CVD) more markedly in women than in men. However, the reported magnitudes of the diabetes-related CVD risk in men and women vary widely between different studies (5). Adverse changes induced by type 2 diabetes in some cardiovascular risk factors, such as HDL cholesterol, triglycerides, LDL, and blood pressure, have been found to be more pronounced in women than in men (6). It is possible that gender may alter the effect of some cardiovascular risk factors in diabetic subjects, leading to a stronger

2016

risk effect in women. Diabetes in women may also interfere more with protective mechanisms in the vascular wall and thereby lead to enhanced atherogenesis and/or thrombogenesis (7). Over the past decade and a half, 4 meta-analyses have examined the relative risk of fatal cardio vascular disease among men and women with diabetes which is depicted in Table 1.Three of these studies found that women with diabetes have an increased risk for death from CVD, which is significantly greater by approximately 12 times than men with T2DM. The fourth study by Kanaya et al observed that although women with diabetes tend to have increased CVD risk compared to men, the result did not reach significance in their meta-analysis. These data strongly suggest that the females are at a greater risk in to CVD-associated mortality than men.

Table :1 Relative risk of fatal cardio vascular disease among men and women with diabetes First Author Number of Design Age Sample Size Conclusions CHD/CVD Mortality Risk Studies (Years) (No. of Diabetics) Orchard28 8 Prospective, 0 >80 50,499 Standardized mortality ratio for (1995) population CHD/CVD mortality risk in men with based diabetes was 1.97 (95% CI, 1.58Y 2.92) and 2.62 (95% CI, 1.98Y4.00) in women with diabetes (8). Lee (2000) 10 Prospective, 35-80 >75,000 Relative risk of coronary death from cohort diabetes was significantly greater in studies women (2.58; 95% CI, 2.05Y3.26) than men (1.85; 95% CI, 1.47Y2.33) (9) . Kanaya 10 Prospective, 35 - 81 6521 Odds ratio for CHD mortality due to (2002) population diabetes was 2.33 (95% CI, 1.9Y2.8) based for men and 2.92 (95% CI, 2.2Y3.8) for women (10). Huxley 37 Prospective >15 128,719 Relative risk for fatal coronary heart (2006) , cohort disease in patients with diabetes studies compared with no diabetes was significantly greater among women (3.50; 95% CI, 2.70Y4.53) than it was among men (2.06; 95% CI, 1.81Y2.34) (11). Chronic subclinical inflammation has been suggested to be involved in the pathogenesis of type 2 diabetes (12). This hypothesis has been Sowmya Rajashekar et al

supported by several prospective studies showing that subjects who developed type 2 diabetes during the follow-up period have had elevated

www.ijetst.in

Page 4927

IJETST- Vol.||04||Issue||01||Pages 4926-4933||January||ISSN 2348-9480 levels of markers of inflammation such as Creactive protein (CRP) or interleukin (IL)-6 at baseline compared with subjects who did not develop the disease (13). There is a lack of data in this regard even though there is a strong evidence for gender differences in associations between diabetes, obesity, endogenous sex hormones, and inflammation (14). Most of the studies that investigated gender differences concerning inflammatory markers and risk for type 2 diabetes were relatively small and have yielded contradictory results. The Hoorn Study, conducted in the Netherlands, reported a significant association between CRP and incident diabetes in men but not in women (15), the opposite was seen in the Mexico City Diabetes Study (5). Finally, in two studies conducted in subjects of Japanese origin, CRP was significantly associated with incident type 2 diabetes in both genders (16,17). In view of the consequences associated with T2DM and increased number of diabetics in India and also the gender based risk factors, there is a need for data on inflammatory markers, to plan prevention and management of CVD among diabetics. Lack of data in this regard has prompted us to investigate the status of the selected inflammatory markers- Homocysteine, C-reactive protein, lipopreotein (a) and Viamin B12 in relation to the duration of diabetes, which is a first study reporting such data among Indian subjects. Materials & Methods A case control study was undertaken in the year 2013 for a period of 6 months to analyze the inflammatory markers among newly detected (NDM) and known diabetic (DM5) subjects. There was a comparison between control subjects, newly detected and known type 2 diabetes mellitus. Study sample: 50 subjects from different sectors were recruited for the study from the diabetic clinic. Before the initiation of the study, ethical clearance (IHEC-UOM No.89 Ph.D / 2013-14) was taken from the heads of the ethical committee Sowmya Rajashekar et al

2016

of the institution. Permission was sought from the heads of the clinic, to recruit the subjects, for the study. The objective of the study was informed to the subjects who visited the hospital and subjects who volunteered to take part in the study were given the consent form. The inclusion criterion was framed for the selection of subjects and those who met these criteria were selected for the study. The selection criteria was as follows : only subjects with type 2 diabetes mellitus in the age group of 30 to 70 yrs, both male and female, newly detected subjects (within a year) and subjects with diabetes mellitus for > 5years, that is known diabetics were included. Subjects without any other comorbidities/ metabolic disorder and who agreed to participate and provide their consent to share all the required details for the study were selected. The subjects were assessed for their anthropometric measurements; BMI was derived and compared with the ICMR ranges for obesity grades, their waist to hip ratio was calculated to arrive at central obesity values. Blood sample (5 ml) was drawn from each subject after an overnight fast. After collection procedure few drops of blood was taken for estimation of peripheral blood smear and haemoglobin. Later the blood sample was centrifuged at 3500rpm for10 -15min at room temperature to separate the serum for further analysis. After this procedure, the serum was analyzed for glucose, triglycerides, total cholesterol, homocysteine, C - reactive protein lipoprotein (a), vitaminB12. Hcy was estimated by enzymatic assay method, CRP by a latex – enhanced turbidimetric invitro immune assay, Lp(a) by turbilatex and vitamin B12 by immunoassay method (18,19) . Statistical Analysis: The Statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, MedCalc 9.0.1, Systat 12.0 and R environment ver.2.11.1 were used for the analysis of the data and Microsoft word and Excel have been used to generate graphs, tables etc. Descriptive and inferential statistical analysis has been carried out in the present study. Results on continuous

www.ijetst.in

Page 4928

IJETST- Vol.||04||Issue||01||Pages 4926-4933||January||ISSN 2348-9480 measurements are presented on Mean  SD (MinMax) and results on categorical measurements are presented in Number (%). Significance is assessed at 5 % level of significance. Analysis of variance (ANOVA) has been used to find the significance of study parameters between three or more groups of patients. Chi-square/ Fisher Exact test has been used to find the significance of study parameters on categorical scale between two or more groups. Pearson correlation between study variables is done to find the correlation (20,21). Result and Discussion The present study was undertaken to mark the importance of bio markers among the two genders and also to identify the risk of cardio vascular disease in T2DM subjects. A comparison between newly detected and known diabetics was also done to show that as the disease progresses the risk of CVD increases. For this purpose inflammatory bio-markers, Hcy, CRP, Lp (a) and Vit B12 were compared with two groups NDM and DM5. Among the 50 subjects in NDM, 33 of them were males and 17 females. In the DM5 group, 30 were males and 20 were females. The age group of the subjects was 30-60 yrs. A large number of subjects selected for the study had a family history of diabetes, stating that diabetes is heredity. In most of the NDM, diabetes was found when they approached for a general

2016

check up and a few of them experienced symptoms of diabetes and had approached for analysis. The somatic status of the subjects is given in table no 2. Anthropometric measurements height and weight were recorded to mark the obesity grades and WHR to mark the central obesity. The BMI results were compared with the ICMR classification and a large number of subjects were either obese or overweight. The BMI (kg/m2) of the males in the NDM was high 21.42 ± 1.60 and among females it was 20.71± 0.85 with a P value of 0.091+ which was significant. The same trend was found among the DM 5 group also. The BMI (kg/m2) of the males was22.77±2.66 and it females it was 22.15±1.98 and the P value being 0.381. The WHR of the subjects in both the groups followed the same trend as BMI the males had a higher WHR than the females in both the groups. The values of the WHR among the males of both the groups were 0.83 ±0.04 in NDM and 0.82±0.04 in the DM5 subjects and among females it was 0.81 ±0.02 and 0.80 ±0.03 among the NDM and DM5 groups respectively. It was also found that as the disease progresses, the BMI of the subjects also increased marginally but the male trend followed among the groups. This suggests that they did not follow a balanced diet and had a lack of exercise and improper lifestyle habits.

Table :2 Somatic status of the subjects Variables

Male

Female

P value

Newly detected type 2 dm 

Age in years

43.36±7.56

40.71±11.02

0.320



BMI (kg/m2)

21.42±1.60

20.71±0.85

0.091+



WHR

0.83±0.04

0.81±0.02

0.124

Known type 2 dm - >5yrs 

Age in years

60.93±10.25

53.95±10.67

0.025*



BMI (kg/m2)

22.77±2.66

22.15±1.98

0.381



WHR

0.82±0.04

0.80±0.03

0.094+

Sowmya Rajashekar et al

www.ijetst.in

Page 4929

2016

IJETST- Vol.||04||Issue||01||Pages 4926-4933||January||ISSN 2348-9480 Subjects had normal haemoglobin levels in the range 12-16%. The peripheral blood smear which is an improvised test to know the haemoglobin condition among the diabetics was also done to know the type of anaemia. The results indicated normal haemoglobin among majority of the subjects. It is a known fact that all the diabetics have high sugar levels, and as expected all the subjects selected have high glucose levels irrespective of the duration of the disease. The lipid profile of these subjects were also measured and compared with the genders and also over a period of disease progression. The values have been tabulated in table no 3. For examining the lipid profile cholesterol (mg/dl), triglycerides (mg/dl), HDL (mg/dl), LDL (mg/dl) and VLDL (mg/dl) were measured and compared within the groups. It was found that males of the NDM had higher levels than the females. The values of NDM males were 187.06±41.28, 228.00±72.65, 44.91 ±6.19, 96.64±45.34 and 45.42±14.49 respectively and females were 178.59 ±19.09,

216.00 ±82.13, 4.59 ±4.62, 95.94 ±19.49 and 39.94 ±13.11 respectively. Where as a slight change was found among the ND5, where in the cholesterol and LDL levels were higher than the males. Females had a value of 201.25 ±45.23 and 110.75 ±46.13 and males had 190.07 ±49.70 and 103.63 ±38.41 respectively. The other values were high among the males that is, TG was 229.93±94.30, HDL was 45.07 ±4.59 and VLDL was 43.80 ± 16.82 and among females it was 212.60 ±113.03, 45.55 ±4.02 and 37.80 ±15.20 respectively. These results showed varied changes in lipid profile. Most of the subjects had high cholesterol and LDL levels, which gives an impression that the diabetic subjects should follow a healthy diet containing more of good fats like monounsaturated and poly unsaturated fats, which in turn would help them from keeping their lipid levels normal, which is important for all diabetics. The lipid parameters were also compared with the NCEP (22) risk value, where in all the values were high, giving an impression of CVD risk among the subjects.

Table :3 Lipid profile of the subjects Variables

Male

Female

P value

Newly detected type 2 dm 

Total cholesterol

187.06±41.28

178.59±19.09

0.427



TGL

228.00±72.65

216.00±82.13

0.599



HDL

44.91±6.19

43.59±4.62

0.443



LDL

96.64±45.34

95.94±19.49

0.952



VLDL

45.42±14.49

39.94±13.11

0.197

Known type 2 dm - >5yrs 

Total cholesterol

190.07±49.70

201.25±45.23

0.423



TGL

229.93±94.30 212.60±113.03

0.559



HDL

45.07±4.59

45.55±4.02

0.703



LDL

103.63±38.41

110.75±46.13

0.557



VLDL

43.80±16.82

37.80±15.20

0.206

Sowmya Rajashekar et al

www.ijetst.in

Page 4930

2016

IJETST- Vol.||04||Issue||01||Pages 4926-4933||January||ISSN 2348-9480 Inflammation always proceeds with diabetes (23) and inflammatory bio – markers have always been linked with endothelial dysfunction in cardiovascular diseases. The estimation of the biomarkers – Hcy, CRP, Lp (a) and Vit B12 helps in identifying the underlying problems of inflammation pertaining to heart and thereby reduces the complications of CVD in diabetic people (24). The levels of the biomarkers have been compared with the standards (25-27) and the values have been tabulated in table no 4. The biomarkers namely homocysteine (µmol/l), C-reactive protein (mg/l), lipoprotein (a) (mg / dl) and vitamin B12 (pg/ml) were analyzed. It was observed that the Hcy and Lp(a) values were higher in males than the females but CRP and Vitamin B12 values

were higher in women when compared to males. (24.54±12.59, 3.81±1.14, 15.67±14.22 and 280.69±77.03 and among the women it was 17.06 ±5.54, 5.84 ±3.47, 10.55 ±12.89 and 352.35± 238.02 respectively). Among the DM5 all the values of the inflammatory markers were higher in males than in females. (It was 24.59 ±5.13, 4.25 ±0.79, 32.70 ±14.54 and 343.43 ±178.39 and in women, 17.64 ±5.07, 3.68 ±0.51, 24.25 ± 9.40 and 338.40 ± 158.15 respectively). The Vit B12 levels of all the subjects from the groups were all within normal limits, but were higher among the DM5 subjects, but were within the normal levels. Several studies have found a deficiency of Vit B12 levels due to drug reaction metformin.

Table 4: Comparison of Homocystien, CRP, LP (a) and Vit B12 in male and female in three groups of patients studied Variables

Male

Female

P value

24.54±12.59

17.06±5.54

0.024*

Newly detected type 2 dm 

Homocystein



CRP

3.81±1.14

5.84±3.47

0.004**



Lp(a)

15.67±14.22

10.55±12.89

0.220



Vit B12

280.69±77.03

352.35±238.02

0.119

Known type 2 dm - >5yrs 

Homocystein

24.59±5.13

17.64±5.07