Telomerase Activity In Diabetic Patients With Angiopathy

,Ayman El Badawy1, Mohamed Shawky1, Amr M.El Hammady1,Mohamed Elassal1 Ashraf Talaat1, Ahmed Dabour1, Khalid Belal2 Internal Medicine1 and Clinical Pathology2 Departments,Banha University,Egypt

Abstract: Background: Diabetes mellitus (DM) is associated with damage to target organs and premature aging and telomeres serve as a mitotic clock and biological marker of senescence. Aim of study: was to A) evaluate telomerase activity in both type 1 and type 2 diabetic patients with microangiopathy and macroangiopathy, B) to study the possible factors that affect the activity of this enzyme in these patients. Subjects and Methods: Study was carried out on 40 patients from those attending diabetes clinics of Banha University Hospital, they were divided into three groups:Group I: 10 type 1 diabetic patients with angiopathy (4males & 6 females),.Group II: 10 type 2 diabetic patients with angiopathy (3males & 7females),.Group III: 20 apparently healthy age & sex matched volunteers serving as a control group. The following laboratory investigations were performed to all patients: Plasma glucose (fasting and post prandial) , Glycated hemoglobin, Lipogram (Total cholesterol, LDLc, HDLc and Triglycerides),Serum creatinine, Urinary microalbumin and Study of telomerase activity in whole blood. Results: Our results showed: that (HBA1C, microalbumin, creatinine, LDL, TG, TCH, PPBS and FBS) are significantly higher in diabetic patients compared to control group where (telomerase and HDL) are significantly lower in diabetic patients compared to control group. Also, Telomerase activity was significantly low in diabetic patients with HbA1C (≥7%) ,LDL-c (>100mg/dl), HDL-c (<45mg/dl), TG (>150mg/dl), TCH (>200mg/dl), microalbuminuria (>30mg/ml) respctively compared to diabetic patients with HbA1C (<7%) , LDL-c (<100mg/dl), HDL-c (>45mg/dl),TG (<150mg/dl), TCH (<200mg/dl), and normoalbuminuria (<30mg/ml). There was significant relation between telomerase activity and macroangiopathic complications, There was nonsignificant correlation between telomerase activity and each of creatinine level and age in the case group. Statistical analysis showed that 80% of diabetic patients (16 patients) were telomerase negative (lower than cut off value ≤49.20), whereas 20% of diabetic patients (4 patients) were telomerase positive (higher than cut off value > 49.20). Conclusion: it was concluded that telomerase enzyme activity decreased in both type 1 and type 2 diabetic patients with angiopathy, There was a relation between telomerase activity and both micro & macroangiopathic .complications

(Key words: telomerase, diabetic patients, angiopathy (micro- or macroangiopathy

:Introduction telomere- lengthening by telomerase, the size of Diabetes mellitus (D.M) is a syndrome of disordered telomeres decreases with each cell division and thus the metabolism, associated with damage to target organs 4 cells have a finite capacity for replication .Telomere and premature aging1. Usually due to a combination of shortness in humans is emerging as a prognostic marker hereditary and environmental causes, resulting in of disease risk, progression, and premature mortality as 2 5 .abnormally high blood sugar levels (hyperglycemia) in diabetes . It was found that telomerase activity Telomerase enzyme is a ribonucleoprotein maintaining decreases in both type 1 and type 2 diabetic patients and the length of the telomeres ( telomeres serve as a mitotic ,since both types of diabetes differ in several aspects, clock and biological marker of senescence) by adding including pathogenesis, severity, participation of G-rich repeats to the end of the eukaryotic immune system and other biological parameters, the chromosomes. Normal human somatic cells, cultured in difference in telomerase activity between them may be vitro, have a strictly limited proliferative potential 6 of interest .The exact mechanism of low telomerase undergoing senescence after about (50-70) population activity in diabetic patients is still controversial and may doublings. In contrast, most of the tumor cells have be explained by hyperglycemia, the proliferative stress .unlimited replicative potential 3 imposed on the relevant cells or by the effect of the Telomeres are composed of telomeric DNA and 7 .oxidative and metabolic stress multiple binding proteins that together act as a Apart from replicative senescence, other exogenous .protective cap on the end of chromosomes factors e.g., oxidative stress, hypertension, The length of telomeres is maintained by a dynamic hypercholesterolemia, and hyperglycemia, may be equilibrium between processes that shorten and associated with low telomerase activity and telomere lengthen telomeric DNA. Telomere shortening serves as attrition. Reduced replicative capacity has been a checkpoint for the initiation of cell cycle arrest, which observed in cells harvested from diabetic subjects and leads to cellular senescence (aging) and apoptosis this finding has been correlated with some of the (death). In healthy human somatic cells that lack degenerative complications of poor glucose control in .diabetic subjects8&9 logistic regression begins with an explanation of the This study was carried out on forty patients selected logistic function, which, like probabilities, always takes from those attending diabetes clinics of Banha .on values between zero and one University Hospital. Patients were divided into three :RESULTS groups: Group I: included 10 type 1 diabetic patients Table (1): shows that (HBA1C, microalbumin, with angiopathy(4males & 6females),Group II: creatinine, LDL, TG, TCH, PPBS and FBS) are included 10 type 2 diabetic patients with angiopathy significantly higher in diabetic patients compared to (3males & 7females), Group III: Included 20 control group where (telomerase and HDL) are apparently healthy age & sex matched volunteers significantly lower in diabetic patients compared to serving as a control group.. Any patient without micro- control group. Fig (1): Statistical analysis showed that or macroangiopathic complications was excluded from 80% of diabetic patients (16 patients) were telomerase .the study negative (lower than cut off value ≤49.20), whereas I- All patients were subjected to a thorough history 20% of diabetic patients (4 patients) were telomerase and clinical examination with stress on Mode of positive (higher than cut off value >49.20).Fig (2) therapy, Microangiopathy (nephropathy, retinopathy, shows the value of telomerase in case group & control and neuropathy), Macroangiopathy (coronary, cerebral, group Table (2) and Fig (3,4,5,6,7,8.9,10) shows that and peripheral atherosclerosis) and Duration of there was significant negative correlation between .diabetes telomerase activity and each of HBA1C level, II- Specimen collection: After 12 hours fasting, 10 ml microalbumin level, , LDL level, TG level and TCH venous blood sample was collected from all subjects by level, FBS level, PPBS level, while there was :veni-puncture and divided as follow significant positive correlation between telomerase ml of blood was delivered into a tube containing 1.5 .1 activity and HDL. There was nonsignificant correlation 1.2 mg/ml K3EDTA used for glycosylated hemoglobin between telomerase activity and each of creatinine level .((HBA1C and age in the case group. Table(3) shows that The remainder was transferred into a plain tube, . 2 telomerase activity was significantly low in diabetic allowed to clot at 37°C followed by centrifugation at patients with HbA1C (≥7%) compared to diabetic 3000 r.p.m for 15 minutes. The serum was collected and patients with HbA1C (<7%).Table (4) shows that used for determination of lipogarm, fasting serum telomerase activity was significantly low in diabetic glucose level and determination of telomerase activity patients with microalbuminuria (>30mg/ml) compared (white blood cells are the most readily available source to diabetic patients with normoalbuminuria of normal human cells in which to measure telomerase (<30mg/ml).Table(5) shows that telomerase activity .(activity directly was significantly low in diabetic patients with LDL-c Another blood sample was withdrawn postprandial . 3 (>100mg/dl) compared to diabetic patients with LDL-c .for determination of post prandial serum glucose level (<100mg/dl).Table(6) shows that telomerase activity III- The following laboratory investigations were was significantly low in diabetic patients with HDL-c :performed to every subject (<45mg/dl) compared to diabetic patients with HDL-c A) Fasting and two hours post prandial serum (>45mg/dl). Table (7) shows that telomerase activity glucose (glucose oxidase) and lipogarm (total was significantly low in diabetic patients with TG cholesterol, triglyceride, HDLcholesterol) and serum (>150mg/dl) compared to diabetic patients with TG creatinine (jaffé method) by Dimension RXL MAX (<150mg/dl).Table(8) shows that telomerase activity auto analyser (Siemens Medical Solution Diagnostics, was significantly low in diabetic patients with TCH UL, USA) with estimation of LDL cholesterol by (>200mg/dl) compared to diabetic patients with TCH .(Fridwald equation LDLc= TCH – (TG/5 + HDLc (<200mg/dl). Table (9) shows that there is non B) Glycated hemoglobin (HbA1C): was assessed by significant difference between telomerase activity and BioSystems reagent kit provided by BioSystems, S.A. creatinine level. Table (10) shows that there was non .(Costa Brava 30, Barcelona (SPAIN significant correlation between telomerase activity and C) Microalbumin: was measured by BioSystems duration of diabetes. Table (11): Logistic Order reagent kit provided by Biosystems, S.A. Costa Brava Regression analysis table, it provided the order of .(30, Barcelona (SPAIN values of patient parameters (HBA1C, lipid profile, D) Specific study (Telomerase activity): was assayed microalbumin, Creatinine) for telomerase activity in in mononuclear cells extract using (Telomerase PCR which HBA1C was the most important ELISA PLUS) kit by Photometric Enzyme parameter(score=21.3 & P=<0.001), followed by LDL Immunoassay for Quantitative Determination of (score=20 & P=<0.001), followed by TCH (score=8.23 Telomerase Activity, Utilizing the Telomeric Repeat & P=<0.001), followed by HDL (score=6.55 & Amplification Protocol (TRAP),10, (developed by P=<0.001), then microalbumin (score=5.3 & P=<0.05) .(Roche Applied Science), (Cat. No. 12 013 789 001 but creatinine was non significant. Table(12) shows that :STATISTICAL ANALYSIS there was significant relation between telomerase Data collected throughout history, basic clinical activity and macroangiopathic complications. Mean ± examination, laboratory investigations and outcome SD of telomerase activity in patients with measures coded, entered and analyzed using Microsoft macroangiopathic complications (7coronary Excel software. Data were then imported into atherosclerosis &1cerebral atherosclerosis &3peripheral Statistical Package for the Social Sciences (SPSS atherosclerosis) (N=11, 55%) Vs patients without version 20.0) software for analysis. Data were presented macroangiopathic complications (N=9, 45%) was as range, mean ±SD and number (%). An explanation of .((35±1.6) Vs(75.8±41.4 Fig(1)ROC Curve .Fig(2):Histogram for Telomerase Value in Control and Case group

Table( 2 ):Correlation of Telomerase Activity with HBA1c, Microalbumin, Creatinine,lipid :profile,FBS,PPS and Age in Case Group :Fig(3):Scatter diagram for correlation between Telomerase activity and HBA1c in case group

Fig(4):Scatter diagram for correlation between Telomerase activity and Microalbumin in case .group .Fig(5): Scatter diagram for correlation between Telomerase activity and LDL-C in case group

Fig(6): Scatter diagram for correlation between Telomerase activity and Triglycerides in case .group Fig(7): Scatter diagram for correlation between Telomerase activity and Total Cholesterol in .case group

.Fig(8): Scatter diagram for correlation between Telomerase activity and FBS in case group

.Fig(9): Scatter diagram for correlation between Telomerase activity and PPS in case group Fig(10): Scatter diagram for correlation between Telomerase activity and HDL-C in case .group

Table(3):Comparison of telomerase activity in patients with HBA1C(<7%)versus patients with .(HBA1C>7%

Table(4): Comparison of telomerase activity in patients with Microalbumin(<30mg/ml)versus .(patients with Microalbumin(>30mg/ml Table(5): Comparison of telomerase activity in patients with LDL-C(<100mg/dl)versus patients .(with LDL-C(>100 mg/dl

Table(6): Comparison of telomerase activity in patients with HDL-C(<45 mg/dl)versus patients .(with HDL-C(>45 md/dl

Table(7): Comparison of telomerase activity in patients with Triglycerides(<150mg/dl)versus .(patients TG(>150mg/dl

Table(8): Comparison of telomerase activity in patients with Total Cholesterol(<200 .(mg/dl)versus patients with TC(>200 mg/dl

Table(9): Comparison of telomerase activity in patients with Creatinine(<1.5mg/dl)vrsus .(patients with Creatinine(>1.5mg/dl

Table(10): Correlation between Telomerase activity and Duration of Diabetes(Persons .(correlation .Table(11):Logistic Order Regression

Table(12):Correlation between Telomerase activity in patients with macroangiopathic .complications versus patients without macroangiopathic complications

diabetic patients (4 patients) were telomerase positive, DISCUSSION fig.(1). Among 16 telomerase negative diabetic Telomerase enzyme is a ribonucleoprotein maintaining patients, 9 patients (56.25%) were type 1 D.M and 7 the length of the telomeres ( telomeres serve as a mitotic .patients (43.75%) were type 2 D.M clock and biological marker of senescence) by adding Our work showed that in comparison to control group, G-rich repeats to the end of the eukaryotic telomerase activity was significantly low in diabetic .chromosomes3 patients. This is in agreement with other studies who Activation of telomerase by extracellular factors may reported that telomeres serve as a mitotic clock and have significant implications in activating and 1 .biological marker of senescence mobilizing stem cells, for tissue repair, organ regeneration and anti-aging regimes11. inactivation of Our results showed that the state of glycemic control telomerase has been demonstrated to be critical to stem had a significant impact on the telomerase activity in cell renewal and potentially to incur aging diseases e.g. diabetic patients. This could be inferred from the .type 2 D.M12 :following There are several reasons why we studied telomerase Telomerase activity was significantly low in diabetic .1 activity in diabetic patients:firstly,to gain an objective patients with HbA1C (≥7) compared to diabetic patients measurement of disease activity as regard angiopathy in with HbA1C (<7), table(3 .( type 1 & type 2 patients; and secondly, trying to find a A significant negative correlation was found between .2 .tool for prediction and management of CVD telomerase activity and each of HBA1C level, FBS and Our study was conducted on forty persons: 10 type 1 .(PPBS in diabetic patients, table (2), fig. (3 ,8 , 9 diabetic patients with angiopathy, 10 type 2 diabetic In Logistic Order Regression analysis: HbA1C .3 patients with angiopathy & 20 age and sex matched greatly affected telomerase activity in which HbA1C .apparently healthy volunteers serving as control Score = (21.3), P= (<0.001),table (11), but there was no Our study showed that 80% of diabetic patients (16 relation between telomerase activityand duration of patients) were telomerase negative, whereas 20% of diabetes according to Pearson‟s correlation table (10). confirmed relation between telomerase and aging as So, telomerase activity depends on glycemic control reported in researches in the past two decades that rather than duration of diabetes. This is in agreement points to a link between organismal aging and aging- with other studies who reported that there was inverse related diseases and cellular senescence caused by correlation observed between (leukocyte telomere telomere shortening. Several lines of evidence strongly lengths, telomerase activity) and Hb1AC & glucose suggest that the resulting telomere dysfunction could level (F.B.S & P.P.B.S) influenced by several factors, have a causal role in some aging and aging-related including oxidative stress consequent to glycemic .diseases20 control, seems to confirm the association between Also, in our results we found that there was no relation accelerated telomere attrition, decreased telomerase between telomerase activity and creatinine, tables activity and increased oxidative stress13. About the 70% (2&9&11). This is in agreement with that plasma of elderly Type2DM patients enrolled in this study creatinine concentration is inversely related to GFR but show HbA1C levels higher than 7%, a value indicative GFR can decrease by 50% before plasma creatinine .of a poor glycemic control 13 concentration rises beyond the normal range. Plasma creatinine concentration doubles for each further 50% fall in GFR. So, normal plasma creatinine doesn't necessarily imply normal renal function, and also, raised plasma creatinine doesn't usually indicate Previous studies have shown that Type2DM patients impaired renal function. This may be due to that with well controlled diabetes did not have statistically changes in plasma creatinine can occur independently significant shorter telomeres than controls, denoting the of renal function, due to changes in muscle bulk, thus a protective effect of efficient glycemic control on decrease can occur in starvation, wasting diseases, .damage and premature senescence of these cells 1 immediately after surgery and in patients treated with In our study all cases had microangiopathic .corticosteroids complications and most of them had decreased our results showed that lipid profile (HDL-c, LDL-c, telomerase activity. So, we can conclude that there is T.G, and T.CH) had a significant impact on the relation between telomerase activity and telomerase activity in diabetic patients. This could be microangiopathic complications. Also, there was 11 inferred from the following : cases (55%) had macroangiopathic complications Telomerase activity was significantly low in diabetic.1 (7coronary atherosclerosis & 1cerebral atherosclerosis patients with LDL-c (≥100), TG (>150), TCH (>200), & 3peripheral atherosclerosis). Our results showed that HDL-c (<45) compared to diabetic patients with LDL-c macroangiopathic complications had a significant (<100), TG (<150), TCH (<200), HDL-c (>45), tables impact on telomerase activity in diabetic patients. This .((5,6,7,8 could be inferred from that telomerase activity was A significant negative correlation was found between .2 significantly low in diabetic patients with telomerase activity and (LDL-c, T.G, T.CH) in diabetic macroangiopathic complications (N=11, 55%) .(patients, table (2), fig.(5, 7, 8 compared to diabetic patients without macroangiopathic A significant positive correlation was found between .3 complications (N=9, 45%), table (12). This is telomerase activity and HDL-c in diabetic patients, consistent with other studies who reported that patients .(table (2), fig.(6 with diabetes are at high risk of macro- and In Logistic Order Regression analysis: LDL-c .4 microangiopathic complications development. affected telomerase activity in which Score = (20), P= Macroangiopathic complications in diabetes are (<0.001), HDL-c affected telomerase activity in which .considered to be as symptoms of premature aging. 14 Score = (6.55), P= (<0.001), T.G affected telomerase Our results showed that microalbuminuria had a activity in which Score = (6.56), P= (<0.001), and also, significant impact on the telomerase activity in diabetic = T.CH affected telomerase activity in which Score :patients. This could be inferred from the following P= (<0.001); table (11). This is in agreement ,(8.23) Telomerase activity was significantly low in diabetic.1 with other studies who reported that diminished levels patients with microalbuminuria (≥30) compared to of high-density lipoprotein cholesterol (HDL-C) are diabetic patients with normoalbuminuria (<30), table associated with an increased risk for atherosclerosis21,22. .((4 ,a disease that is marked by chronic, low grade, A significant negative correlation was found between .2 inflammation and increased burden of oxidative telomerase activity and microalbumin in diabetic .stress23,24 .(patients, table (2), fig.(4 It was found that there is positive correlation between In Logistic Order Regression analysis: microalbumin .3 HDL-C and telomerase activity in many25,26. Low levels also, affected telomerase activity in which of HDL cholesterol are considered as one of the .(microalbumin Score = (5.3), P= (<0.05), table (11 components of „atherogenic dyslipidemia25and low This is in agreement with other studies who reported HDL cholesterol phenotypes have been recently shown that some Type2DM complications, such as to display elevated oxidative stress, attenuated microalbuminuria (a urinary albumin excretion rate .antioxidative activity27and accelerated senescence28 >30 mg/24 h.) and MI, are associated with lower .telomerase activity &shorter telomere length ,15,16,17,18,19 Also, in our results we found that there was no relation :Conclusions .(between telomerase activity and age, tables (1 & 2 From the previous results it was concluded that This may be due to narrow age range in our study with telomerase enzyme activity decreased in both type 1 and (Mean ± SD) = (39.6±5.2) years. Although there is Kim NW, Piatyszek MA, Prowse KR,-(10 type 2 diabetic patients with angiopathy. There was a Harley CB, West MD, Ho PLC, et al., strong and significant relation between telomerase activity and the occurrence and presence of both micro (1994): Specific association of human & macroangiopathic complications. Telomerase activity telomerase activity with immortal cells and and telomere length may be a possible new marker in .cancer. Science; 266: 2011-2015 the prediction of cvd and diabetes representing the Sarin KY, Cheung P, Gilison D, Lee E,-(11 contribution of tissue aging , atherosclerosis and their Tennen RI, Wang E, et al., (2005): linked pathology, also we recomend that this trial must Conditional telomerase induction causes be done on a large number in diabetic patients to . assess , compare and confirm these results proliferation of hair follicle stem cells. Nature; .436: 1048–1052 Lee HW, Blasco MA, Gottlieb GJ,-(12 REFERENCES Horner JW, Greider CW and DePinho RA Uziel O, Singer JA, Danicek V, Sahar G,-(1 (1998): Essential role of mouse telomerase in Berkov E, Luchansky M, et al., (2007): highly proliferative organs. Nature; 392: 569– Telomere dynamics in arteries and .574 mononuclear cells of diabetic patients: effect Yamada (2003): Telomere shortening,-(14 of diabetes and of glycemic control. atherosclerosis and metabolic syndrome. .Experimental Gerontology; 42: 971–978 .Intern Med; 42: 135-136 Chan JC (2009): Diabetes in Asia:-(2 Tentolouris N, Nzietchueng R and-(15 Epidemiology, risk factors, and Cattan V (2007): White blood cells telomere .pathophysiology. JAMA; 301: 2129-2145 length is shorter in males with type 2 diabetes Fuster J and Andrés V (2006): Laboratory-(3 and microalbuminuria. Diabetes Care; 30: of Vascular Biology. C/Jaime; 99: 1167-1180. .2909–2915 .doi: 10.1161 Fitzpatrick AL, Kronmal RA, Gardner-(16 Liew CW, Holman A and Kulkarni RN-(4 JP, Psaty BM, Jenny NS, Tracy RP, et al., (2009): The roles of telomeres and telomerase (2007): Leukocyte telomere length and cardiovascular disease in the cardiovascular health study. American Journal of in beta‐cell regeneration. Diabetes Obes .Epidemiology; 165:14–21 Farzaneh-Far R, Cawthon RM and Na-(17 B (2008): Prognostic value of leukocyte telomere length in patients with stable .Metab; 4: 21‐29 coronary artery disease. Arterioscler Thromb .Vasc Biol; 28:1379–1384 Spyridopoulos I, Erben Y and-(18 Karlseder J (2003): Telomere repeat-(5 Brummendorf TH (2008): Telomere gap binding factors: keeping the ends in check. between granulocytes and lymphocytes is a .Cancer Lett; 194: 189–197 determinant for hematopoetic progenitor cell Adaikalakoteswari A (2009): Telomere-(6 impairment in patients with previous shortening occurs in Asian Indian type 2 myocardial infarction. Arterioscler Thromb .diabetes. Diabetes Med; 22: 1151-1156 .Vasc Biol; 28: 968–974 Richter T and Von Zglinicki T (2007): - (7 Wilson WR, Herbert KE and Mistry Y-(19 A continuous correlation between oxidative (2008): Blood leukocyte telomere DNA stress and telomere shortening in fibroblasts. content predicts vascular telomere DNA .Exp Gerontol; 42: 1039–1042 content in humans with and without vascular The Diabetes Control and - (8 disease. European Heart Journal; 29: 2689– Complications Trial Research Group .2694 (1993): The effect of intensive treatment of Dokal I and Vulliamy T (2003): - (20 diabetes on the development and progression Dyskeratosis congenita: its link to telomerase of long-term complications in insulin .and aplastic anaemia. Blood Rev; 17:217–225 dependent diabetes mellitus. N Eng J Med; Assmann G, Schulte H, von Eckardstein-(21 .329: 977–986 A, and Huang Y (1996): High-density Ritz E and Orth SR (1999): Nephropathy-(9 lipoprotein Cholesterol as a predictor of in-patients with type 2 diabetes mellitus. N. .Eng. J. Med.; 341 1127–1133 AdaikalakoteswariA,-(26 coronary heart disease risk. Atherosclerosis; Balasubramanyam M, Ravikumar R, Deepa .124: 11–20 R, and Mohan V (2007): Association of Nicholls SJ, Tuzcu EM and Sipahi I-(22 telomere shortening with impaired glucose (2007): Statins, high-density lipoprotein tolerance and diabetic macroangiopathy. cholesterol and regression of coronary .Atherosclerosis; 195:83–89. doi:10.1016 .atherosclerosis. JAMA; 297: 499–508 Kontush A, de Faria EC, Chantepie S-(27 Hansson GK and Libby P (2006): The-(23 and Chapman MJA (2005): immune response in atherosclerosis: a double- Normotriglyceridemic, low HDL-cholesterol .edged sword. Nat Rev Immunol; 6:508–519 phenotype is characterized by elevated Gutierrez J, Ballinger SW, Darley--(24 oxidative stress and HDL particles with Usmar VM and Landar A (2006): Free attenuated antioxidative activity. radicals, mitochondria, and oxidized lipids: the .Atherosclerosis; 182: 277–285 emerging role in signal transduction in Nofer J, Walter M and Assmann G-(28 .vascular cells. Circ Res; 99:924–932 (2005): Current understanding of the role of Misra A, Luthra K and Vikram NK-(25 high-density lipoproteins in atherosclerosis (2004): Dyslipidemia in Asian Indians: and senescence. Exp Rev Cardiovasc Ther; 3: determinants and significance. J Assoc; 52: .1071–1086 .137–142