HOSPITAL UNIVERSITÁRIO PEDRO ERNESTO SERVIÇO DE DIABETES E METABOLOGIA TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
Eu, ___________________________________________________, aceito participar da pesquisa “Avaliação da reatividade microvascular e da rigidez arterial em pacientes diabéticos tipo 1: correlação com variáveis clínicas e laboratoriais”, realizada pela Dra.
Alessandra Saldanha de Mattos Matheus, na Disciplina de Diabetes, orientada pela Prof.
Dra Marília de Brito Gomes e co-orientada pelo Professor visitante Eduardo Tibiriçá (telefones 2587-6324 ou 2598-4451 ramal 219) por minha livre e espontânea vontade, podendo haver recusa ou retirada do consentimento sem penalização , e prejuízo do meu tratamento no ambulatório de Diabetes, estando garantidos meu sigilo e privacidade.
Estou ciente de que serei submetido a realização de exame capilaroscópico da pele de dedos das mãos, exame de fluxometria laser Doppler com iontoforese de acetilcolina e nitroprussiato na pele do antebraço, além de verificação do índice de resistência das artérias conforme descrito abaixo:;
1- O exame de fluxometria laser Doppler é realizado através da aplicação de eletrodos na pele do antebraço, sendo percebida apenas uma sensação de formigamento durante alguns minutos no local da pele em contato com o eletrodo;
2- Será também colocado um aparelho na extremidade do dedo médio de uma das mãos para verificação da onda de pulso, sendo indolor;
3- Responderei corretamente a um questionário, e permitirei que realizem meu exame físico;
4- Permitirei a coleta do meu sangue com material esterilizado e descartável, através da punção de uma veia no meu membro superior;
5- Poderá ocorrer a formação de uma mancha roxa no local de retirada do sangue (hematoma);
6- Serei comunicado sobre o resultado dos meus exames caso estejam alterados ou se assim desejar;
7- Serei encaminhado ao Ambulatório de Diabetes deste hospital na presença de alguma alteração no exame físico e/ou laboratorial para fins de acompanhamento adequado se desejar.
Rio de Janeiro, ____ de ________________ de ______ Pesquisador Rio de Janeiro, ___de ________________de ________ Pesquisado Responsável (pacientes com idade <19 anos; crianças e adolescentes)
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Short Communication
Evaluation of microvascular endothelial function in patients with type 1 diabetes using laser-Doppler perfusion monitoring: Which method to choose?
Marilia B. Gomesa, Alessandra S.M. Matheusa, Eduardo Tibiriçáa,b,⁎
aDiabetes Unit, State University of Rio de Janeiro, Brazil
bLaboratory of Neuro-Cardiovascular Pharmacology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil
a b s t r a c t
The evaluation of microvascular function is essential in the investigation of the pathophysiology of cardiometabolic diseases [Struijker-Boudier, H.A. et al., 2007. Evaluation of the microcirculation in hypertension and cardiovascular disease. Eur. Heart J. 28, 2834–2840]. In clinical research and practice, the study of microcirculation is of great value in the assessment of the effects of medical interventions and monitoring disease progression. It is well-known that patients with type 1 and 2 diabetes have microvascular dysfunction that results from numerous factors including hyperglycemia, oxidative stress and insulin resistance [Schalkwijk, C.G., Stehouwer, C.D., 2005. Vascular com-plications in diabetes mellitus: the role of endothelial dysfunction. Clin. Sci. (Lond). 109, 143–159]. Moreover, skin microvascular dysfunction in type 1 diabetes precedes symptoms of end-organ microvascular disease [Khan, F. et al., 2000. Impaired skin microvascular function in children, adolescents, and young adults with type 1 diabetes. Diabetes Care 23, 215–220]. In this study, we assessed skin microvascular function of patients with type 1 diabetes using laser-Doppler perfusion monitoring (LDPM) coupled with physiological and pharmacological local vasodilator stimuli.
© 2008 Elsevier Inc. All rights reserved.
Research design and methods
This cross-sectional study included 50 outpatients with type 1 diabetes (21 males) aged 32.8± 1.66 years with a disease duration of 15 ± 1.3 years, followed up at a public hospital and 46 control subjects without underlying diseases (22 males) matched for age (±5 years), gender, BMI and smoking habits. The study was approved by the local Ethics Committee and participants gave written informed consent. Studies were performed in the morning after a 20-minute rest in the supine position in a temperature-controlled room (23 ± 1°C), approximately 2h after a light breakfast. Patients took their usual dose of morning insulin. Endothelium-dependent and -independent vasodilation of skin micro-circulation was evaluated with an LDPM system in combination with iontophoresis (Periflux 5001 and PeriIont, Perimed, Järfälla, Sweden) of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, for noninvasive and continuous measurement of skin microvascular perfusion changes (perfusion units, PU = 10mV). Drug delivery electrodes were incorporated into the head of the laser probe and the probe temperature was standardized to 32°C in order to avoid variations in skin temperature, and, consequently, in the measurements of microvascularflow. The drug delivery electrodes werefilled with 200µl of 1% ACh (Sigma Chemical CO, USA) and were attached with the laser probe to a standardized site of the forearm. The dispersive electrode was attached about 15cm away from the electrophoresis chamber. After registration of restingflux for 5min, four doses of ACh were delivered using an anodal current (0.1mA for 10, 20, 40 and 80s; total charges of 1, 2, 4 and 8mC) with 120-second intervals. Variability of the responses to ACh between and within subjects has been shown to be ~ 20% (Morris and Shore, 1996; Newton et al., 2001).
Using a new delivery electrode applied to a different location on the same forearm, four doses of 1% SNP (Sigma Chemical CO, USA) were delivered using a cathodal current (same charges and intervals as for ACh). During the post-occlusive reactive hyperemia (PORH) test, arterial occlusion was performed with a suprasystolic pressure using a sphygmo-manometer for 3min (biological zero) applied to the arm of the subjects. Following the
release of pressure, maximumflux, time to maximumflux (TM), time to half recovery after hyperemia (TH2) and area under the curve of PORH were measured. Finally, to investigate maximal skin microvascular vasodilation, we used prolonged (20min) local heating of a laser probe to 44°C. The use of the area under the curve in the assessment of skin microvascular reactivity using LDPM is well-validated, since it represents the overallflux response to different physiological and pharmacological stimuli (Leslie et al., 2003; Opazo Saez et al., 2005; Rossi et al., 2006).
Data are presented as median (25th–75th percentile) since values do not follow a Gaussian distribution (Shapiro–Wilk normality test). Group differences for dose–
response curves were analyzed after logarithmic transformation using two-way ANOVA for repeated measures followed by Bonferroni posttests; other data were tested using nonparametric tests (Mann WhitneyUtest). The number of patients and controls gave 80% power to detect differences of ~ 35% in vascular responses.Pvaluesb0.05 defined statistical significance.
Results
Mean resting flux did not differ between control subjects and patients with type 1 diabetes (Table 1). Microvascular responses to both ACh and SNP were significantly reduced in patients, either when expressed in PU (Table 1) or cutaneous vascular conductance (mV/
mmHg, data not shown). Maximal percentage increase influx was lower in patients, when compared to controls, either during ACh [643.6 (266.5–1064) vs. 1111(616.4–1446), Pb0.001] or SNP [694.3(358.1– 906.9) vs. 791.1(477.1–1392),Pb0.05] stimulation. On the other hand, maximal skin microvascular vasodilation induced by thermal hyper-emia was not significantly different between patients and controls (PN0.05,Table 1). During PORH, maximal increase influx and area under the hyperemic response did not differ significantly between patients and controls, but the time to reach maximumflux and the Microvascular Research 76 (2008) 132–133
⁎Corresponding author. Av. Brasil 4365, 21045-900, Rio de Janeiro, Brazil.
E-mail address:etibi@ioc.fiocruz.br(E. Tibiriçá).
0026-2862/$–see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.mvr.2008.04.003
Contents lists available atScienceDirect
Microvascular Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m v re
time to half recovery after hyperemia were higher in patients than in controls (Pb0.05,Table 1).
Conclusions
The present study confirmed that both endothelium-dependent (ACh-iontophoresis) and -independent (SNP-(ACh-iontophoresis) skin microvascular vasodilator responses are reduced in adult patients with type 1 diabetes, as described previously in children and adolescents with type 1 diabetes (Khan et al., 2000). Even though microvascular response to ACh-ion-tophoresis is considered to be a specific test for endothelial function (Newton et al., 2001), this response has already been attributed to a complex interaction of factors including nitric oxide, prostacyclin and even activation of sensory nerves (Barac et al., 2007; Cracowski et al., 2006).
Conversely, microvascular vasodilation induced by PORH, which is also considered to be, at least in part, endothelium (shear stress)-mediated (Meredith et al., 1996), was similar between control subjects and patients with type 1 diabetes. PORH responses have already been shown to be reduced (Shore et al., 1991) or not (Golster et al., 2005; Wilson et al., 1992) in patients with type 1 diabetes. These conflicting results could be ac-counted for by the use of different experimental protocols, small groups of patients (differences in age, gender and duration of diabetes) or could eventually be related to the repeatability of the methods. In order to avoid variations in skin temperature, and, consequently, in the measurements of skin microvascularflow and to reduce inter-individual variations, we used
standardized room and probe temperatures (Cracowski et al., 2006;
Hansell et al., 2004). In the present study we decided to clamp the tem-perature of the probe to a thermoneutral temtem-perature (32°C). It is unlikely that this procedure could have influenced the results, since skin tem-perature of study subjects, measured before clamping of the probe temperature, is around 30°C. Thus, temperature of the probe was in-creased only slightly in order to standardize recording conditions. It is also noteworthy that PORH has already been suggested to be dependent (Binggeli et al., 2003; Meredith et al.,1996) or independent (Gooding et al., 2006; Wong et al., 2003) of nitric oxide and prostanoid release. In our study, the only difference between patients and controls, concerning PORH response, was the longer time that patients took to reach peakflux during hyperemic response. Finally, in contrast to previous observations (Khan et al., 2000), in our study skin maximal hyperemic response induced by local heating was not significantly different between patients and controls. Interestingly, it has recently been shown that this latter pheno-menon is also dependent on nitric oxide bioavailability (Gooding et al., 2006).
In conclusion, our study showed that different methodologies designed to assess skin microvascular endothelial function generate dissimilar results in patients with type 1 diabetes. Thus, the use of a single physiological or pharmacological stimulation coupled to LDPM should be avoided.
Acknowledgments
This investigation was supported by grants of FAPERJ (Fundação de Amparo à Pesquisa, Rio de Janeiro, Brazil) and CNPq (Conselho Nacional de Desenvolvimento Tecnológico, Brasília, Brazil).
References
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Golster, H., et al., 2005. Impaired microvascular function related to poor metabolic control in young patients with diabetes. Clin. Physiol. Funct. Imaging. 25, 100–105.
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possible mechanisms. J. Vasc. Res. 43, 270–277.
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Khan, F., et al., 2000. Impaired skin microvascular function in children, adolescents, and young adults with type 1 diabetes. Diabetes Care 23, 215–220.
Leslie, S.J., et al., 2003. Validation of laser Dopplerflowmetry coupled with intra-dermal injection for investigating effects of vasoactive agents on the skin microcirculation in man. Eur. J. Clin. Pharmacol. 59, 99–102.
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Table 1
Comparison of skin microvascular reactivity between control subjects and patients with type 1 diabetes
Acetylcholine-mediated increases influx(perfusion units)
Doses (mC) Control subjects Type 1 diabetes
Restingflux 3.2 (2.2–5.5) 3.38 (1.8–4.6)
1 7.5 (3.8–13.1) 7.3 (3.3–11.4)
2 15.6 (9.5–29.8) 13.6 (5.3–21.1)
4 30.9 (19.6–44.4) 23.4 (38.4–9.8)⁎
8 39.5 (28.9–71.2) 32.9 (16.7–50.4)⁎⁎⁎
AUC (PU/s) 7701 (4959–11604) 4819 (2298–9208)⁎⁎
Sodium nitroprusside-mediated increases influx (perfusion units)
Restingflux 3.2 (2.4–5.3) 3.3 (2.4–4.7)
1 6.96 (4.2–12.3) 6.0 (3.6–9.9)
2 10.25 (5.0–19.1) 8.5 (5.8–15.3)
4 19.0 (10.1–39.1) 15.5 (8.5–27.0)
8 49.5 (26.4–77.1) 35.9 (22.5–52.5)⁎
AUC (PU/s) 9103 (5588–17106) 7423 (4517–9891)⁎
Post-occlusive reactive hyperemia
Variable Control subjects Type 1 diabetes
Restingflux 3.6 (2.8–5.8) 3.3 (2.6–4.9)
Maximumflux (PU) 19.4 (12.5–27.1) 17.7 (10.6–23.7)
AUC (PU/s) 400.1 (190.3–747.5) 383.0 (201.0–589.6)
TM (s) 11.2 (6.7–15.5) 13.2 (9.3–23.7)⁎
TH2 (s) 21.7 (16.7–29.0) 26.8 (17.6–35.7)⁎
Thermal hyperemia
Maximumflux (PU) 67.8 (41.5–99.22) 51.6 (43.0–67.4) Dose–response curves (total chargers in millicoulombs, mC) for endothelium-dependent (acetylcholine) and -inendothelium-dependent (sodium nitroprusside) vasodilators were expressed in arbitrary perfusion units (PU) and areas under the curves (AUC) in PU/s. TM, time to maximumflux during hyperemia; TH2, time to half recovery after PORH. Data are expressed as median (25th–75th percentile) since values do not follow a Gaussian distribution (Shapiro–Wilk normality test). Group differences for dose–
response curves were analyzed after logarithmic transformation using two-way analysis of variance followed by Bonferroni posttests; other data were tested using nonparametric tests (Mann WhitneyUtest).
*Pb0.05; **Pb0.01; ***Pb0.001, when compared control subjects.
M.B. Gomes et al. / Microvascular Research 76 (2008) 132–133 133