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1 Evolution of target organ damage and hemodynamic parameters over years in patients with increased insulin resistance. The LOD-DIABETES trial. Journal: BMJ Open Manuscript ID bmjopen Article Type: Research Date Submitted by the Author: 0-Oct-0 Complete List of Authors: Gomez-Marcos, Manuel Recio-Rodriguez, Jose; La Alamedilla Health Center, Research Unit, Patino-Alonso, Maria; Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla, Agudo-Conde, Cristina; Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla,, Research Unit Rodriguez-Sanchez, Emiliano; Univ Salamanca, Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla, Maderuelo-Fernandez, Jose; Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla, Castilla and León Health Service (SACYL). Network of Preventive Activities and Health Promotion in Primary Care (REDIAPP) Gomez-Sanchez, Leticia; Primary care research unit La Alamedilla, Gomez-Sanchez, Marta; Primary care research unit La Alamedilla, GarciaOrtiz, Luis; La Alamedilla health centre. University of Salamanca, Primary care research unit La Alamedilla <b>primary Subject Heading</b>: Diabetes and endocrinology Secondary Subject Heading: Cardiovascular medicine Keywords: Type diabetes mellitus, Metabolic syndrome, Target organ damage, Arterial stiffness, Drug treatment BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

2 Page of BMJ Open Title: Evolution of target organ damage and hemodynamic parameters over years in patients with increased insulin resistance. The LOD-DIABETES trial. Manuel Ángel Gómez-Marcos*,,, José Ignacio Recio-Rodríguez,, María Carmen Patino-Alonso,, Cristina Agudo-Conde,, Emiliano Rodríguez-Sanchez,,, Jose Angel Maderuelo-Fernandez,, Leticia Gómez-Sánchez, Marta Gomez-Sanchez and Luís García-Ortiz,,, LOD-DIABETES Group.. Primary Care Research Unit, the Alamedilla Health Center. Salamanca, Spain. Castilla and León Health Service SACYL. REDIAPP: Research Network on Preventive Activities and Health Promotion. Biomedical Research Institute of Salamanca (IBSAL). Salamanca, Spain. Medicine Department, University of Salamanca, Salamanca, Spain.. Statistics Department, University of Salamanca, Salamanca, Spain.. Biomedical and diagnostic sciences department, University of Salamanca, Salamanca, Spain. LOD-DIABETES Group. REDIAPP: Research Network on Preventive Activities and Health Promotion, Spain. Correspondence to*: Manuel Angel Gómez Marcos, Primary Care Research Unit, the Alamedilla Health Center Avda. Comuneros, 00 - Salamanca, Spain. Tel: + ; fax + ; magomez@usal.es addresses: MAG: magomez@usal.es JIR: donrecio@gmail.com MCP: carpatino@usal.es CA: cagudoconde@yahoo.es ER: emilianorodriguezsanchez@yahoo.es JAM: jmaderuelo@saludcastillayleon.es LG: leticiagmzsnchz@gmail.com MG: magoma@usal.es LG: lgarciao@usal.es Number of words: Tables: Figures: References: BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

3 Page of Title: Evolution of target organ damage and hemodynamic parameters over years in patients with increased insulin resistance. The LOD-DIABETES trial. ABSTRACT The purpose of this study was to evaluate the evolution of the cardiac, renal and vascular target organ damage (TOD) as well as the hemodynamic parameters in patients with type diabetes or metabolic syndrome (MetS) over four years of follow-up. Methods: We performed a prospective observational study involving patients ( with type diabetes and with MetS) who were followed for years. Measurements were blood pressure, blood glucose, lipids, smoking, body mass index (BMI), waist circumference, HOMA-Ir, hs-c-reactive protein and fibrinogen levels. We also evaluated vascular (carotid intima-media thickness (IMT), pulse wave velocity (PWV) and ankle/brachial index (ABI)), heart (Cornell voltage-duration product and Sokolow) and renal target organ damage (creatinine, glomerular filtration and albumin/creatinine index). The hemodynamic parameters were central (CAIx) and peripheral (PAIx) augmentation index. Results: In the year, subjects with type diabetes increased IMT thickness. These patients had more number of plaques and IMT> 0.0 mm. In subjects with MetS, we only found an increase in the number of plaques. We found no changes in PWV, CAIx and PAIx. The diabetic patients increased the percentage of vascular TOD. There were no differences in renal or cardiac TOD nor in the percentage of TOD in the MetS patients. Conclusions: This prospective study showed that the evolution of vascular TOD is different in subjects with type diabetes respect to those with MetS. While IMT and PWV increased in type diabetes, these were not modified in MetS. The renal and cardiac TOD evolution, as well as the PAIx and CAIx, did not change in either group. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

4 Page of BMJ Open Trial Registration: Clinical Trials.gov Identifier NCT00 Key words: Type diabetes mellitus. Metabolic syndrome. Target organ damage. Arterial stiffness. Drug treatment. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

5 Page of INTRODUCTION Cardiovascular disease morbidity-mortality is greater in people with type diabetes or metabolic syndrome (MetS). The presence of target organ damage (TOD) increases the risk of cardiovascular complications independently of the existing estimated risk. In this context, left ventricular hypertrophy (LVH) assessed according to electrocardiographic criteria increases the risk of coronary complications and stroke. The worsening of renal function assessed by increased creatinine levels decreases the estimated glomerular filtration rate (egfr) or may increase urine albumin creatinine ratio. These are independent risk factors that increase the cardiovascular morbidity and mortality in patients with type diabetes. Peripheral arterial disease evaluated by the ankle/brachial index (ABI) is correlated to the development of coronary complications, the incidence of stroke, and cardiovascular mortality. Ultrasound measurements of common carotid artery intima-media thickness (IMT) allow the evaluation of vascular structure and the early detection of atherosclerotic lesions, which represents a good predictor of future vascular events and is a surrogate marker of atherosclerosis. The mean estimates of IMT progression ranged from 0.00 to 0.00 mm per year for common carotid artery intima-media thickness. 0 IMT in type diabetes is 0. mm greater than in the controls. This implies an age increment of 0 years, and is associated with a 0% increase in cardiovascular risk. Likewise, an increase in arterial stiffness as assessed by pulse wave velocity (PWV) and central and peripheral augmentation index (CAIx and PAIx) predicts future cardiovascular events and mortality from any cause in both hypertensive subjects and in the general population. - Thus, it is important to understand the evolution of the cardiac, renal and vascular TOD, hemodynamic parameters, as well as antihypertensive, lipid-lowering and antidiabetic drugs during the study period. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

6 Page of BMJ Open The purpose of this study was to evaluate the evolution of the cardiac, renal and vascular TOD as well as the hemodynamic parameters in patients with type diabetes or metabolic syndrome (MetS) over four years of follow-up. METHODS: Study design A prospective observational study was carried out in the primary care setting with a follow up of four years. This study analyzed subjects who were included in the LOD-DIABETES study (NCT00). Study population: Using consecutive sampling, we included patients who visited their Family Physician between January 00 and January 00 with type diabetes (n = ) as defined by the American Diabetes Association criteria or MetS (n = ) defined according to the National Cholesterol Education Program, ATP III. Exclusion criteria included patients unable to comply with the protocol requirements (psychological and/or cognitive disorders, failure to cooperate, educational limitations and problems in understanding written language, failure to sign the informed consent document). Patients participating or programmed to participate in a clinical trial during the study were also excluded, as were patients with serious comorbidities representing a threat to life over the subsequent months. The sample sized was estimated to detect statistically significant differences in carotid IMT greater than or equal to 0.0 mm between baseline and years. We used an alpha risk of 0.0 and a beta risk of 0. in a two-sided test. We further assume a standard deviation of 0. mm, based in previous studies to show that subjects are necessary. We anticipated a drop-out rate of %. An independent ethics committee of BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

7 Page of health area of Salamanca (Spain) approved the study. All participants gave written informed consent according to the general recommendations of the Declaration of Helsinki. MEASUREMENT: A detailed description has been published elsewhere on how the clinical data were collected including anthropometric measurements, blood pressure, TOD assessment. Blood pressure: Three measurements of systolic (SBP) and diastolic blood pressure (DBP) were collected with a validated OMRON model M sphygmomanometer (Omron Health Care, Kyoto, Japan). We used the average of the last two according to the recommendations of the European Society of Hypertension. 0 Vascular assessment: Carotid femoral pulse wave velocity (PWV) and peripheral (PAIx) and central augmentation index (CAIx): These parameters were estimated using the SphygmoCor System (AtCor Medical Pty Ltd., Head Office, West Ryde, Australia). Pulse wave velocity (PWV) was estimated with patients in the supine position. The pulse wave of the carotid and femoral arteries were analyzed to estimate the delay with respect to the ECG wave and calculate the PWV. Distance measurements were collected with a measuring tape from the sternal notch to the carotid and femoral arteries at the sensor location. A TOD was identified if the PWV was higher than m/sec. The central augmentation index (CAIx) is a composite index that integrates the amount of the wave that is reflected back to the aorta depending on the tone of the resistance arteries, which are the main peripheral reflecting sites. Using the above system (Px Pulse Wave Analysis) with the patient in the sitting position and resting the arm on a BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

8 Page of BMJ Open rigid surface, pulse wave analysis was performed with a sensor in the radial artery. A mathematical transformation then estimated the aortic pulse wave. The reliability of these measurements was evaluated before the study using the CAIx intra-class correlation coefficient, which showed values of 0. (% CI: 0. to 0.) for intraobserver agreement on repeated measurements. According to the Bland-Altman analysis, the mean difference for intra-observer agreement (% limits of agreement) was 0. (-. to 0.). From the morphology of the aortic wave, the CAIx was estimated using the following formula: increase in central pressure 00/pulse pressure. The value was adjusted to a heart rate of by the SphygmoCor System device. The peripheral augmentation index (PAIx) is a measurement taken directly from the late systolic shoulder of the peripheral arterial waveform. It is the ratio of the difference in amplitude between the second peak and the diastolic pressure over the difference between the first peak and diastolic pressure. The PAIx was calculated as (second peak SBP (SBP) - DBP)/(first peak SBP - DBP) x 00, to yield a percent (%) value. Assessment of vascular structure by carotid intima media thickness (IMT): Carotid ultrasound to assess carotid IMT was performed by two investigators trained for this purpose before starting the study. The reliability of such recordings was evaluated before the study, using the intra-class correlation coefficient, which showed values of 0. (% CI: 0. to 0.) for intra-observer agreement on repeated measurements in 0 subjects, and 0.0 (% CI: 0. to 0.) for inter-observer agreement. According to the Bland-Altman analysis, the mean difference for inter-observer agreement (% limits of agreement) was 0.0 (-0.0 to 0.0). A Sonosite Micromax ultrasound (Sonosite Inc., Bothell, Washington, USA) device paired with a 0 MHz multifrequency high-resolution linear transducer with Sonocal software was used for BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

9 Page of automatic measurements of IMT to optimize reproducibility. Measurements were made of the common carotid after examining a 0 mm longitudinal section cm from the bifurcation. Measurements were performed at the proximal and distal wall in the lateral, anterior and posterior projections. They followed an axis perpendicular to the artery to discriminate two lines one for the intima-blood interface and the other for the mediaadventitious interface. A total of measurements were obtained for the right carotid and measurements for the left carotid. We used the average values (average IMT) that was automatically calculated by the software. The measurements were obtained with the subject lying down, with the head extended and slightly turned opposite of the carotid artery under study. Average IMT values were considered abnormal if > 0.0 mm or if there were atherosclerotic plaques with a diameter of. mm or a focal increase of 0. mm or 0% of the adjacent IMT. 0 Evaluation of peripheral artery involvement: This was assessed using the ankle-brachial index (ABI) and was calculated in the morning for patients who had not drank coffee or smoked tobacco for at least h prior to the measurement. The room temperature was C. Patients were supine with the feet uncovered. The pressure in the lower limbs was measured after resting for 0 min using a portable Watch BP Office for assessing the ABI (Microlife AG Swiss Corporation Espenstrasse ; CH- Widnau/Switzerland). The ABI was calculated automatically for each foot by dividing the higher of the two systolic pressures in the ankle by the higher of the two systolic pressures in the arm. An ABI < 0. was considered abnormal. 0 Renal assessment: Kidney damage was assessed by measuring creatinine plasma concentration and glomerular filtration rate (egfr) as estimated according to the Modification of Diet in BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

10 Page of BMJ Open Renal Disease-Isotopic Dilution Mass Spectrometry (MDRD-IDMS). Proteinuria was assessed by the albumin/creatinine ratio following the 0 European Society of Hypertension/European Society of Cardiology Guidelines criteria. TOD was defined as plasma creatinine of. mg per 00 ml or higher in men and. mg per 00 ml or higher in women as well as an egfr below 0 ml per min or albumin/creatinine ratio 0 mg/g. 0 Cardiac assessment: The electrocardiographic examination was performed using a General Electric MAC.00 ECG System (General Electric, Niskayuna, NY, USA) that automatically measures the voltage and duration of waves and estimates the criteria of the Cornell voltage duration product (Cornell VDP) and Sokolow Lyon product. The TOD was defined according to the 0 European Society of Hypertension/European Society of Cardiology Guidelines criteria. 0 The individuals performing the different tests were blinded to the clinical data. All assessments were made within 0 days. Statistics Continuous variables were expressed as mean ± standard deviation and qualitative variables used a frequency distribution. We analyzed the evolution of quantitative variables at follow-up with repeated measures analyses by GLM procedure corrected by the Bonferroni method. We considered the presence or absence of sphericity and performed the Greenhouse and Geisser correction. The IMT and PWV were analyzed with repeated measures analyses unadjusted and adjusted for age, gender, office mean blood pressure and atherogenic index. We used the Cochran tests to contrast the hypothesis of two or more related proportions. The data were analyzed using the SPSS BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

11 Page 0 of version 0.0 statistical package (SPSS Inc., Chicago, Illinois, USA). A value of p < 0.0 was considered statistically significant. RESULTS Throughout the first year of follow up, two males died as result of acute myocardial infarction, one with type diabetes and other with MetS (aged and years, respectively). Subsequently, two non-fatal cardiovascular events occurred in the type diabetes group. In the MetS group, there was a cerebrovascular event, and subjects developed type diabetes. The flow chart is shown in Figure. The mean age was ± years (0 ± years in diabetics and ± years in MetS). The frequency of males was.% (.% in diabetics,.% in MetS). Table shows the cardiovascular risk factors, biochemical data and drugs analyzed in each of the four evaluations in subjects with type diabetes and MetS. The mean time of evolution of type diabetes was. ±. years. Table shows the annual assessments of vascular, renal and cardiac TOD in patients with type diabetes and MetS. In subjects with Type diabetes, we observed an increase in IMT, the number of plaques and the percentage of subjects with IMT> 0. mm in the th year. There were also changes in the ABI (p< 0.0) and in the Sokolow criteria. In subjects with MetS there were changes in the number of subjects with mean maximum IMT > 0. mm, subjects with plaques (p = 0.0) in the ABI (p = 0.00) and the Sokolow and Cornell PDV criteria. The average IMT mean increased 0.00 mm in diabetic patients and 0.00 mm in subjects with MetS per year (p = 0.0). PWV increased 0. m/sec in diabetic patients and decreased 0. m/sec in subjects with MetS per year (p = 0.00). 0 BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

12 Page of BMJ Open Figure shows the trend and the percentage of patients with type diabetes or Mets with vascular, renal and cardiac TOD in each of the four measurements. The most prevalent TOD in the both groups is the carotid artery injury (% in type diabetes and % in MetS) in the last assessment. In subjects with Type diabetes, the percentage of patients with overall and vascular TOD increased (p <0.0). There were no differences in renal or cardiac TOD. In MetS patients, we found no changes in the TOD. In the unadjusted repeated measures analyses, we found differences in IMT (p = 0.00), but not in the PWV (p = 0.0; Figure ). After adjusting for age, gender, mean blood pressure and atherogenic index, the differences in the four-year of follow-up of the IMT disappear (p = 0.0), but did not modify the PWV (p = 0.) and some interactions remain. DISCUSSION: This study included a -year follow-up period of patients with type diabetes or with MetS, and it showed an increase of carotid IMT and PWV TOD higher in type diabetes than MetS. There were not significant differences in the frequency of renal and cardiac TOD in Type diabetes. Subjects with MetS have not any significant increase of TOD. These results are consistent with the meta-analysis of Lorenz MW et al. (based on strokes from studies). 0 This study concluded that the association between IMT progression evaluated with ultrasounds, cardiovascular risk and the risk of subsequent cardiovascular events in the general population remains unproven. 0 In subjects with type diabetes, Tripolt NJ et al. conducted an intensive intervention on different cardiovascular risk factors over two years and managed to reduce the IMT, BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

13 Page of but none of the markers including endothelial function parameters, were useful in predicting such changes. In contrast to these results, the Multi Ethnic Study of Atherosclerosis (based on strokes) had a significant and positive association between yearly mean IMT progression and risk of stroke. Baldassarre et al. concluded that rapid progression of IMT was associated with adverse cardiovascular outcome. The women and men with MetS of the Tromsø study had higher levels of IMT at follow up than those without MetS. MetS predicted IMT progression in people 0 years of age and younger, but not in other age groups, which indicated that MetS may be involved in the initiation of the atherosclerotic process. In stepwise multivariable analyses with each component of the MetS entered separately and adjusted for age, LDL-cholesterol, and smoking, we found that only impaired glucose tolerance was associated with progression of IMT in men. In a post hoc analysis, on hypertensive patients in the European Lacidipine Study on Atherosclerosis (ELSA), 0 IMT progression was slightly greater in patients with MetS, but this was not significant after adjusting for other cardiovascular risk factors. Only type diabetes patients showed increases in PWV m / sec during the monitoring period. Both, age and blood pressure, are the two most important determinants of PWV in the general population as well as in type diabetes and MetS patients. PWV progression was the same in both groups because of age differences. Our findings suggest that patients with increased insulin resistance did not have differences in CAIx and PAIx. These results are consistent with previously published data in a Chinese population. Similarly, an intensive, multifactorial treatment of screen-detected diabetes during years in a general practice showed a significant impact on aortic stiffness, whereas effects on the other hemodynamic measurements, BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

14 Page of BMJ Open augmented pressure and augmentation index are smaller and not statistically significant. The behavior of ABI was similar in both patient groups both in terms of absolute ABI values and the percentage of patients with ABI <0.. Similar data have been published by Ito et al. in patients with type diabetes. However, it must be remembered that in diabetic patients the standard threshold sensitivity (0.) is lower and thus the efficiency of ABI is limited. Moreover, in this group of patients the sensitivity for values between 0.-. is low (-%). Finally, our results are in line with those published in the Hoorn Study, which concluded that the associations between ABI and cardiovascular and all-cause mortality were similar in individuals with and without diabetes. The evaluation of left ventricular hypertrophy based on the Cornell voltage-duration product and Sokolow criteria showed differences between the four measurements conducted in patients with MetS, but did not show a clear trend. The same applies to Sokolow criteria in subjects with type diabetes, but there were no variables that explain these changes. We found no differences between renal function nor the percentage of subjects with renal TOD in the two groups. This is likely justified because up to 0% of subjects with type diabetes and micro albuminuria do not progress to clinical proteinuria or even spontaneously reverse. This may partly explain the results of this study. Likewise, the declining blood pressure seen in MetS patients would prevent the progression of nephropathy. This study has some limitations that must be considered. First, the number of subjects per group limits the power of analysis. Furthermore, these patients were not randomized, but involved consecutive sampling. The two groups are not fully balanced in terms of age ( years of difference), which may influence the course. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

15 Page of This prospective study showed that the evolution of vascular TOD is different in subjects with type diabetes respect to those with MetS. While IMT and PWV increased in Type diabetes, these were not modified in MetS. The renal and cardiac TOD evolution, as well as the PAIx and CAIx, did not change in either group. Acknowledgments We are grateful to all professionals participating in the LOD-DIABETES study. Coordinating Center: Manuel A Gómez-Marcos. La Alamedilla Health Center (Castilla y León Health Service-SACYL, Salamanca, Spain) Cristina Agudo-Conde, Leticia Gomez-Sanchez, Marta Gomez-Sanchez, Carmen Castaño-Sanchez, Carmela Rodriguez-Martin, Benigna Sanchez-Salgado, Angela de Cabo Laso, Emiliano Rodriguez-Sanchez, Jose Angel Maderuelo-Fernandez, Emilio Ramos-Delgado, Carmen Patino-Alonso, Jose I Recio-Rodriguez, and Luis Garcia- Ortiz. Authors contributions MAGM designed the study, wrote the protocol, participated in fund raising, interpreted the results, prepared the manuscript draft, performed all analytical testing, interpreted the results and reviewed the manuscript. and corrected the final version of the manuscript. JIRR and CAC participated in the study design, data collection and manuscript review. LGS, MGS, ERS and JAMF participated in the study design, interpretation of results and manuscript review. MCPA: participated in the analysis of results and final review of the manuscript. LGO participated in the protocol design, fund raising, analysis of results and final review of the manuscript. All authors reviewed and approved the final version of the manuscript. Sources of Funding: The project has been funded by the Institute of Health Carlos III, (ISCIII) of the Ministry of Economy and Competitiveness (Spain) through the Network BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

16 Page of BMJ Open for Prevention and Health Promotion in Primary Care (rediapp, RD/000), cofinanced with European Union ERDF funds and the Autonomous Government of Castilla and León in 00 and 0 (GRS. /A/0; GRS /B/), and the Intensification of Research Program. Competing interests The authors declare that they have no competing interests. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

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19 Page of Baldassarre D, Veglia F, Hamsten A, Humphries SE, Rauramaa R, de Faire U, et al. Progression of carotid intima-media thickness as predictor of vascular events: results from the IMPROVE study. Arterioscler Thromb Vasc Biol 0;():-.. Herder M, Arntzen KA, Johnsen SH, Mathiesen EB. The metabolic syndrome and progression of carotid atherosclerosis over years. The Tromso study. Cardiovasc Diabetol 0;:. 0. Zanchetti A, Hennig M, Baurecht H, Tang R, Cuspidi C, Carugo S, et al. Prevalence and incidence of the metabolic syndrome in the European Lacidipine Study on Atherosclerosis (ELSA) and its relation with carotid intima-media thickness. J Hypertens 00;():-0.. Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D, et al. Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J 00;():-0.. Stehouwer CD, Henry RM, Ferreira I. Arterial stiffness in diabetes and the metabolic syndrome: a pathway to cardiovascular disease. Diabetologia 00;():-.. Zhang M, Bai Y, Ye P, Luo L, Xiao W, Wu H, et al. Type diabetes is associated with increased pulse wave velocity measured at different sites of the arterial system but not augmentation index in a Chinese population. Clin Cardiol 0;(0):-.. Johansen NB, Charles M, Vistisen D, Rasmussen SS, Wiinberg N, Borch-Johnsen K, et al. Effect of intensive multifactorial treatment compared with routine care on aortic stiffness and central blood pressure among individuals with screendetected type diabetes: the ADDITION-Denmark study. Diabetes Care 0;():0-.. Ito H, Komatsu Y, Mifune M, Antoku S, Ishida H, Takeuchi Y, et al. The estimated GFR, but not the stage of diabetic nephropathy graded by the urinary albumin excretion, is associated with the carotid intima-media thickness in patients with type diabetes mellitus: a cross-sectional study. Cardiovasc Diabetol 00;:.. Dachun X, Jue L, Liling Z, Yawei X, Dayi H, Pagoto SL, et al. Sensitivity and specificity of the ankle--brachial index to diagnose peripheral artery disease: a structured review. Vasc Med 00;():-.. Potier L, Abi Khalil C, Mohammedi K, Roussel R. Use and utility of ankle brachial index in patients with diabetes. Eur J Vasc Endovasc Surg 0;():0-.. Hanssen NM, Huijberts MS, Schalkwijk CG, Nijpels G, Dekker JM, Stehouwer CD. Associations between the ankle-brachial index and cardiovascular and all-cause mortality are similar in individuals without and with type diabetes: nineteenyear follow-up of a population-based cohort study. Diabetes Care 0;():-.. Ito H, Mifune M, Abe M, Oshikiri K, Antoku S, Takeuchi Y, et al. Hypertension resistant to antihypertensive agents commonly occurs with the progression of diabetic nephropathy in Japanese patients with type diabetes mellitus: a prospective observational study. BMC Nephrol 0;:. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

20 Page of BMJ Open Figure legend: Figure. Study flow chart. Represents the subjects analyzed each year. Cardiovascular events in each group and the evolution of patients with metabolic syndrome to Type diabetes. Cerebrovascular (CV). MetS: Metabolic syndrome, DM: Type diabetes. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

21 Page 0 of Figure. Changes between the four years of follow-up in target organ damage (TOD). a: TOD in type diabetes. b: TOD in Metabolic syndrome. IMT: Intima Media Thickness, cf-pwv: carotid femoral Pulse Wave Velocity, ABI: Ankle Brachial Index, GFR: Glomerular Filtration Rate, ACR: Albumin Creatinine Ratio. In type diabetes: p <0.00 in TOD overall, TOD vascular, carotid and cf-pwv. 0 BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

22 Page of BMJ Open Figure. Estimated unadjusted means (figure a and b), and adjusted by age, gender, atherogenic index and office blood pressure (figure c and d) of intima-media thickness (IMT) and pulse wave velocity (PWV) in type diabetes and in metabolic syndrome patients. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

23 Page of Table. Changes in cardiovascular risk factors and medications used in diabetes mellitus and metabolic syndrome patients. Type diabetes mellitus patients st year(n=) nd year(n=) rd year(n=) th year(n=) p value Smoker s n (%) (.) (.0) (.) (.) 0. Ischemic heart disease n (%) (.) (.) (.) (.) 0. Cerebrovascular disease n (%) (.) (.) (.0) (.0).000 BMI (kg/m ) 0.±.0.±..±..±. 0. Waist circumference (cm) 0.±. 0.±. 0.±. 0.±. 0. Total Cholesterol (mg/dl).±.0.±..±..±. 0.0 Tryglicerides (mg/dl).±..±.0.±..±0. 0. LDL cholesterol (mg/dl) 0.±. 0.0±. 0.±. 00.± HDL cholesterol (mg/dl).±..±..±. 0.±. 0.0 Serum glucose (mg/dl).±..±..±..±. 0. HbAc (%).±..0±..0±.0.±.0 0. HbAc (mmol / mol) ±. ±. ±. ±. 0. HOMA-IR.±..0±.0.±..±. 0.0 hs-c-reactive protein ( mg/dl) 0.±0. 0.±0. 0.±0. 0.±0. 0. Fibrinogen (mg/dl).±..±. 0.±..±. 0.0 Office SBP (mm Hg).±..±..±..±. 0. Office DBP (mm Hg) <0.0 Mean Antihypertensive Drugs.±..0±..±..±. 0.0 Antihypertensive Drugs n (%) (.) (.) (.) (.) 0. Mean Lipid lowering drugs 0.0±0. 0.0±0. 0.±0. 0.± Lipid lowering drugs n (%) (.) (.) (.) (.) 0. Mean antidiabetic drugs.±0..±0..±0..± Antidiabetic drugs n (%) (.) (.0) (.) (.0) 0. Metabolic syndrome patients st year (n=) nd year (n =) rd year (n=) th year (n=) p value Smoker s n (%) (0.) (0.) (.) (.) 0.00 Ischemic heart disease n (%) (.) (.) (.) (.).000 Cerebrovascular disease n (%) 0 (0.0) 0 (0.0) 0 (0.0) (.) 0. BMI (kg/m ).±. 0.±..±. 0.±. 0. Waist circumference (cm) 0.±. 0.±.0 0.±. 0.±. 0.0 Total Cholesterol (mg/dl).0±. 0.±. 0.0±.0.±. 0.0 Triglycerides(mg/dL).±..0±..±0..±. 0. LDL cholesterol (mg/dl) 0.±. 0.±..0±..±. 0.0 HDL cholesterol (mg/dl).±.0.±. 0.±..±0. <0.0 Serum glucose (mg/dl).±..±. 0.±..±. 0.0 HbAc (%).±0..±0..±0..±0. 0. HbAc (mmol / mol) ±.0 ±. ±. ±. 0.0 HOMA-IR *.±..±.0.±..± hs-c-reactive protein ( mg/dl) 0.±0. 0.±0. 0.±0. 0.± Fibrinogen (mg/dl).±..±..±..± Office SBP (mm Hg) *.±..0±..±..±. <0.0 Office DBP (mm Hg).±..±0..±0..±. <0.0 Mean Antihypertensive Drugs * 0.±0.0.±.0.±..±0. <0.0 Antihypertensive Drugs n (%) * 0 (.) (.) 0 (.) (.) <0.0 Mean Lipid lowering drugs 0.0±0. 0.±0. 0.±0. 0.± Lipid lowering drugs n (%) (0.) (.) 0 (.) (.) 0.00 Data for qualitative variables are expressed as n: number (%) and quantitative variables as mean ± standard deviation. BMI: Body Mass Index; LDL: Low Density Lipoprotein; HDL: High Density Lipoprotein; HbAC: Glycosylated Hemoglobin; HOMA-IR: Homeostasis Model Assessment Insulin Resistance; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure. * p<0.0 between st and nd year; p<:0.0 between st and rd year; p<0.0 between st and th year. p<0.0 between nd and rd year; p<0.0 between nd and th year p<0.0 between rd and th year. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

24 Page of BMJ Open Table. Changes in target organ damage and arterial stiffness in type diabetes mellitus and metabolic syndrome patients. Type diabetes mellitus patients st year(n=) nd year(n=) rd year(n=) th year(n=) p value Vascular Carotid IMT average (mm) 0.±0. 0.±0.0 0.±0. 0.0±0. <0.0 Carotid IMT average > 0.0 mm n (%) (.) (.0) (.0) 0(.) 0.0 Carotid IMT maxima average (mm) 0.±0. 0.±0. 0.±0. 0.±0. <0.0 Carotid IMT maxima average > 0.0 mm n (%) 0 (.) (.) (0.) (.) 0.0 Plaques carotid n (%) (.) (.) 0 (.) (.) 0.0 ABI *.±0..0±0..±0..±0. <0.0 PWV (m/s).±..±.0.±. 0.±. 0. CAIx 0.±..±. ±..±. 0. PAIx.0±.00.±..±..±. 0. Renal Serum creatinine (mg/dl) 0.±0. 0.±0. 0.±0.0 0.± egfr(ml/min/. m) 0.0±..0±0.0.±0..± Albumin/creatinine ratio (mg/g).±..±..±. 0.±. 0. Heart Cornell VDP (mm/ms).±..±..±..±.0 0. Sokolow (mm/ms).±.0.±.0 0.±..0± Metabolic syndrome patients st year(n=) nd year(n=) rd year(n=) th year(n=) p value Vascular Carotid IMT average (mm) 0.±0. 0.±0. 0.±0.0 0.±0. 0. Carotid IMT average > 0.0 mm n (%) (.) (.) (.) (.0) 0.00 Carotid IMT maxima average (mm) 0.±0. 0.±0. 0.±0. 0.± Carotid IMT maxima average > 0.0 mm n (%) (.) (.) (.) (.) 0.0 Plaques carotid n (%) (.0) (.0) (.) (.) 0.0 ABI.±0..±0.0.0±0..± PWV (m/s).±.0.±..0±.0.± CAIx.±.00.±0..±..±. 0.0 PAIx.±. 0.±0.0.0±..0±. 0. Renal Serum creatinine, (mg/dl) 0.±0. 0.0±0. 0.±0. 0.±0. 0. egfr(ml/min/. m).±.0.±..±. 0.±. 0. Albumin/creatinine Ratio (mg/g).±..±..±0..0±. 0. Heart Cornell VDP (mm/ms) *.±..±..±..± Sokolow (mm/ms) 0.±..±..±..±. <0.0 Data for qualitative variables are expressed as n: number and (%) and quantitative variables as mean ± standard deviation. IMT: Intima Media Thickness Carotid; ABI: Ankle Brachial Index; PWV: Pulse Wave Velocity; CAIx: Central Augmentation Index; PAIx: Peripheral Augmentation Index; egfr: Estimated Glomerular Filtration Rate; Cornell VDP: Cornell Voltage Duration Product. * p<0.0 between st and nd year; p<:0.0 between st and rd year; p<0.0 between st and th year. p<0.0 between nd and rd year ; p<0.0 between nd and th year. p<0.0 between rd and th year. BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

25 Page of Figure. Study flow chart. Represents the subjects analyzed each year. Cardiovascular events in each group and the evolution of patients with metabolic syndrome to Type diabetes. Cerebrovascular (CV). MetS: Metabolic syndrome, DM: Type diabetes. 0xmm (00 x 00 DPI) BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

26 Page of BMJ Open Figure. Changes between the four years of follow-up in target organ damage (TOD). a: TOD in type diabetes. b: TOD in Metabolic syndrome. IMT: Intima Media Thickness, cf-pwv: carotid femoral Pulse Wave Velocity, ABI: Ankle Brachial Index, GFR: Glomerular Filtration Rate, ACR: Albumin Creatinine Ratio. In type diabetes: p <0.00 in TOD overall, TOD vascular, carotid and cf-pwv. 0x00mm (00 x 00 DPI) BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

27 Page of Figure. Estimated unadjusted means (figure a and b), and adjusted by age, gender, atherogenic index and office blood pressure (figure c and d) of intima-media thickness (IMT) and pulse wave velocity (PWV) in type diabetes and in metabolic syndrome patients. 0x00mm (00 x 00 DPI) BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

28 Evolution of target organ damage and hemodynamic parameters over years in patients with increased insulin resistance. The LOD-DIABETES. Prospective observational study. Journal: BMJ Open Manuscript ID bmjopen r Article Type: Research Date Submitted by the Author: -Jan-0 Complete List of Authors: Gomez-Marcos, Manuel Recio-Rodriguez, Jose; La Alamedilla Health Center, Research Unit, Patino-Alonso, Maria; Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla, Agudo-Conde, Cristina; Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla,, Research Unit Rodriguez-Sanchez, Emiliano; Univ Salamanca, Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla, Maderuelo-Fernandez, Jose; Salamanca Institute for Biomedical Research. (IBSAL)., Primary care research unit La Alamedilla, Castilla and León Health Service (SACYL). Network of Preventive Activities and Health Promotion in Primary Care (REDIAPP) Gomez-Sanchez, Leticia; Primary care research unit La Alamedilla, Gomez-Sanchez, Marta; Primary care research unit La Alamedilla, Garcia-Ortiz, Luis; La Alamedilla health centre. University of Salamanca, Primary care research unit La Alamedilla <b>primary Subject Heading</b>: Diabetes and endocrinology Secondary Subject Heading: Cardiovascular medicine Keywords: Type diabetes mellitus, Metabolic syndrome, Target organ damage, Arterial stiffness, Drug treatment BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

29 Page of BMJ Open Title: Evolution of target organ damage and hemodynamic parameters over years in patients with increased insulin resistance. The LOD-DIABETES. Prospective observational study. Manuel Ángel Gómez-Marcos*,,, José Ignacio Recio-Rodríguez,, María Carmen Patino-Alonso,, Cristina Agudo-Conde,, Emiliano Rodríguez-Sanchez,,, Jose Angel Maderuelo-Fernandez,, Leticia Gómez-Sánchez, Marta Gomez-Sanchez and Luís García-Ortiz,,, LOD-DIABETES Group.. Primary Care Research Unit, the Alamedilla Health Center. Salamanca, Spain. Castilla and León Health Service SACYL. REDIAPP: Research Network on Preventive Activities and Health Promotion. Biomedical Research Institute of Salamanca (IBSAL). Salamanca, Spain. Medicine Department, University of Salamanca, Salamanca, Spain.. Statistics Department, University of Salamanca, Salamanca, Spain.. Biomedical and diagnostic sciences department, University of Salamanca, Salamanca, Spain. LOD-DIABETES Group. REDIAPP: Research Network on Preventive Activities and Health Promotion, Spain. Correspondence to*: Manuel Angel Gómez Marcos, Primary Care Research Unit, the Alamedilla Health Center Avda. Comuneros, 00 - Salamanca, Spain. Tel: + ; fax + ; magomez@usal.es addresses: MAG: magomez@usal.es JIR: donrecio@gmail.com MCP: carpatino@usal.es CA: cagudoconde@yahoo.es ER: emilianorodriguezsanchez@yahoo.es JAM: jmaderuelo@saludcastillayleon.es LG: leticiagmzsnchz@gmail.com MG: magoma@usal.es LG: lgarciao@usal.es Number of words: Tables: Figures: References: BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.

30 Page of Title: Evolution of target organ damage and hemodynamic parameters over years in patients with increased insulin resistance. The LOD-DIABETES Prospective observational study. ABSTRACT The purpose of this study was to evaluate the evolution of the cardiac, renal and vascular target organ damage (TOD) as well as the hemodynamic parameters in patients with type diabetes or metabolic syndrome (MetS) over four years of follow-up, as well as to analyze the differences between both groups. Methods: We performed a prospective observational study involving patients ( with type diabetes and with MetS) who were followed for years. Measurements were blood pressure, blood glucose, lipids, smoking, body mass index (BMI), waist circumference, HOMA-Ir, hs-c-reactive protein and fibrinogen levels. We also evaluated vascular, carotid intima-media thickness (IMT), pulse wave velocity (PWV) and ankle/brachial index (ABI), heart (Cornell voltage-duration product and Sokolow) and renal target organ damage (creatinine, glomerular filtration and albumin/creatinine index). The hemodynamic parameters were central (CAIx) and peripheral (PAIx) augmentation index. Results: In the year, subjects with type diabetes increased IMT thickness. These patients had more number of plaques and IMT> 0.0 mm. In subjects with MetS, we only found an increase in the number of plaques. We found no changes in PWV, CAIx and PAIx. The diabetic patients increased the percentage of vascular TOD. There were no differences in renal or cardiac TOD nor in the percentage of TOD in the MetS patients. Conclusions: This prospective study showed that the evolution of vascular TOD is different in subjects with type diabetes respect to those with MetS. While IMT and BMJ Open: first published as 0./bmjopen on June 0. Downloaded from on January 0 by guest. Protected by copyright.