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Circulation. 1997;95:618-625

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(Circulation. 1997;95:618-625.)
© 1997 American Heart Association, Inc.

Articles

Hyperreactivity to Nitrovasodilators in Forearm Vasculature Is Related to Autonomic Dysfunction in Insulin-Dependent Diabetes Mellitus

Sari Makimattila, MD; Matti Mantysaari, MD; Per-Henrik Groop, MD; Paula Summanen, MD; Antti Virkamaki, MD; Anna Schlenzka, MD; Johan Fagerudd, MD; Hannele Yki-Jarvinen, MD

the Department of Medicine, Divisions of Endocrinology and Diabetology (S.M., A.V., A.S., H.Y.-J.), Nephrology (P.-H.G., J.F.), and Ophthalmology (P.S.), Helsinki (Finland) University Central Hospital, and Research Institute of Military Medicine, Central Military Hospital (M.M.), Helsinki, Finland.

Correspondence to Hannele Yki-Jarvinen, MD, The University of Texas Health Science Center at San Antonio, Department of Medicine/Diabetes Division, 7703 Floyd Curl Dr, San Antonio, TX 78284. E-mail jarvinen@arwen.uthscsa.edu.


*    Abstract
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 *Abstract
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 down arrowResults
 down arrowDiscussion
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Background The link between diabetes and vascular disease is poorly understood. Data regarding endothelial function in vivo in patients with insulin-dependent diabetes mellitus (IDDM) have been inconsistent with in vitro studies demonstrating hyperglycemia-induced impairments in endothelium-dependent vasodilation.

Methods and Results We determined whether alterations in neural control of the vascular tone might contribute to blood flow responses to intrabrachial infusions of acetylcholine (ACh), sodium nitroprusside (SNP), and L-N-monomethyl-arginine (L-NMMA) in 22 men with IDDM (12 with normoalbuminuria, HbA1c=8.6±0.3%; 10 with macroalbuminuria, HbA1c=8.6±0.3%) and 11 matched normal men. Autonomic function was assessed from reflex vasoconstriction to cold, the blood pressure response to standing and hand grip, and heart rate variation, including spectral analysis, during controlled breathing, and the Valsalva maneuver. IDDM with macroalbuminuria exhibited hyperresponsiveness to both ACh and SNP compared with the patients with normoalbuminuria or normal subjects. Reflex sympathetic vasoconstriction to cold was severely impaired in the IDDM patients with macroalbuminuria (-19±6%) compared with normoalbuminuric patients (-39±5%, P<.05) and normal subjects (-54±7%, P<.001). The macroalbuminuric patients also had evidence of autonomic dysfunction during controlled and deep breathing tests and during the Valsalva maneuver. Within the group of IDDM patients, neither the urinary albumin excretion rate nor other parameters such as HbA1c or serum cholesterol correlated with forearm blood flow during the vasoactive drug infusions. There were, however, significant inverse correlations between several measures of both sympathetic and parasympathetic autonomic functions and vascular hyperresponsiveness to SNP and ACh. For example, the Valsalva ratio was inversely correlated with the increase in blood flow in response to infusion of 3 (r=-.74, P<.001) and 10 (r=-.73, P<.001) µg/min SNP and 7.5 (r=-.73, P<.001) and 15 (r=-.75, P<.001) µg/min ACh.

Conclusions These data are consistent with idea that altered neurotransmission is an important determinant of vascular reactivity of diabetic blood vessels to nitrovasodilators in vivo.


Key Words: endothelium • vasodilation • glucose • vessels


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Endothelial dysfunction, measured as the blood flow response to NO synthesis–dependent vasoactive drugs, characterizes patients at risk for developing atherosclerosis, such as those with hypercholesterolemia.1 Exposure of isolated arterial segments to chronic hyperglycemia in vitro2 3 4 5 also impairs endothelium-dependent vasodilation. These defects have been suggested to be mediated by increased activation of protein kinase C,3 increased generation of oxygen-derived free radicals,4 and quenching of NO by advanced glycosylation end products5 and may contribute to the increased prevalence of atherosclerosis in IDDM.6

Diabetic nephropathy is the most important cause of the excessive cardiovascular mortality of patients with IDDM.7 Excessive cardiovascular mortality in patients with IDDM is caused by sudden death resulting from cardiac arrhythmias, autonomic neuropathy,8 9 and premature atherosclerosis.6 10 The latter data suggest that endothelium-dependent vasodilation is more severely impaired in patients with IDDM and albuminuria than in those with normoalbuminuria or in normal subjects, provided that no other factors determine vascular reactivity.

In vivo studies addressing endothelial function in IDDM have yielded contradictory results. ACh-induced endothelium-dependent vasodilation has been mostly normal11 12 13 14 or, in some studies, impaired.15 16 Similarly, the blood flow response to the endothelium-independent vasodilator SNP has been mostly normal,11 13 14 15 16 although it was impaired in one study.12 The reasons for the variable results are unclear but could be related to the presence or absence of microvascular or macrovascular complications, sex, ambient glucose concentrations during the endothelial function tests, and differences in glycemic control.13

Vascular tone in patients with IDDM not only is controlled by endothelial NO synthesis but also is influenced by the autonomic nervous system.17 Sympathetic denervation increases blood flow and is in its extreme form characterized by the warm diabetic neuropathic foot and the development of Charcot arthropathy.18 Sympathetic denervation also decreases reflex vasoconstriction to such stimuli as cold.19 It is unclear to what extent alterations in neural blood flow control might modulate the blood flow responses to endothelium-dependent and endothelium-independent vasoactive agents. In none of the studies addressing endothelial function in patients with IDDM has the degree of autonomic dysfunction or its possible influence on vascular function been considered. Furthermore, no previous study included patients with overt diabetic nephropathy, a condition known to markedly increase the risk of cardiovascular mortality.7

The present study was undertaken to determine whether endothelium-dependent vasodilation is altered in patients with overt diabetic nephropathy who were carefully characterized with respect to the presence of autonomic dysfunction. The patients with nephropathy were compared with IDDM patients with normoalbuminuria and normal subjects. Our results suggest that autonomic dysfunction is an important determinant of vascular reactivity in patients with complicated IDDM.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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 down arrowDiscussion
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Subjects
Twenty-two men with IDDM and 11 normal men volunteered for the studies. The diabetic patients were recruited from the outpatient clinic on the basis of the following criteria: (1) age between 18 and 50 years, (2) age at diagnosis of diabetes <30 years, and (3) undetectable fasting C-peptide concentration (<0.1 nmol/L). A history and physical examination were obtained and laboratory tests were performed in all subjects to exclude diseases other than IDDM. All patients and normal subjects had normal blood counts, electrolyte concentrations, liver enzymes, thyroid functions, and ECGs (data not shown). Three timed overnight urine collections were performed to classify the patients according to their UAERs (Table 1Down). Normoalbuminuria was defined as a UAER <20 µg/min; macroalbuminuria was defined as a rate >200 µg/min in at least two of three consecutive overnight urine samples.20 Twelve patients had normoalbuminuria; 10 had macroalbuminuria. These groups were matched with each other and the normal subjects with respect to age, body mass index, and composition (Table 1Down). The duration of IDDM was significantly longer in patients with macroalbuminuria than normoalbuminuria. Patients with macroalbuminuria had a significantly higher blood pressure and serum creatinine concentration than those with normoalbuminuria (Table 1Down). None of the IDDM patients had symptoms of ischemic heart disease or diabetic autonomic or other neuropathy. To grade diabetic retinopathy, two 45° fundus color photographs were taken of each eye through dilated pupils and classified by an ophthalmologist (Dr Summanen) in a blinded fashion.21 22 Eight patients with IDDM with macroalbuminuria and 1 with normoalbuminuria had severe nonproliferative or proliferative retinopathy; the rest had mild or moderate nonproliferative retinopathy. The diabetic patients were treated with 2 (n=1), 3 (n=3), or 4 (n=16) injections of a combination of intermediate- and short-acting insulins. Two patients were using continuous subcutaneous insulin infusion therapy. Table 1Down gives the mean insulin doses. Seven patients with IDDM with macroalbuminuria were treated with ACE inhibitors. This medication was discontinued for 2 days before the forearm blood flow measurements. Informed written consent was obtained after the purpose, nature and potential risks were explained to the subjects. The experimental protocol was designed and performed according to the principles of the Helsinki Declaration and was approved by the Ethical Committee of the Helsinki University Central Hospital.


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  		Table 1. Characteristics of the Study Groups

Protocol
All subjects were studied on two separate occasions with at least a 1-week interval between the studies. On one occasion, in vivo endothelial function was determined by measuring the effects of intra-arterial infusions of endothelium-dependent and endothelium-independent vasodilators on forearm blood flow. On the other occasion, a set of autonomic function tests was performed to assess autonomic neuropathy. In these tests, the rapid heart rate changes induced by deep breathing at a given pace, an increase and release of intrathoracic pressure, and a change in body posture were considered to reflect parasympathetic function. The increase in blood pressure in response to isometric muscular contraction, such as hand grip, and the maintenance of blood pressure in the head-up position are thought to reflect sympathetic control of blood vessel tone (vasoconstriction). Heart rate changes during and after the Valsalva maneuver also reflect sympathetic vasoconstrictor function. For 2 days before the studies, the subjects ingested a weight-maintaining diet containing at least 200 g/d carbohydrates with 15% to 20%, 45% to 50%, and 35% to 40% of calories from protein, carbohydrate, and fat, respectively.

Forearm blood flow responses to endothelium-independent (SNP) and endothelium-dependent (ACh and L-NMMA) vasoactive agents were determined. To avoid possible confounding effects of acute hyperglycemia on endothelial function,3 an intravenous infusion of insulin (0.1 mU·kg-1·min-1) was started at 7:30 AM after a 10- to 12-hour overnight fast to normalize the plasma glucose concentration in the diabetic patients. Insulin was infused and blood samples were drawn through a 18-gauge (Venflon, Viggo-Spectramed) catheter inserted into the right antecubital vein. Normoglycemia was reached within 163±15 minutes after the insulin infusion was begun. Glucose was infused if necessary to maintain normoglycemia on the basis of plasma glucose measurements performed at 20-minute intervals. The plasma glucose concentration averaged 5.7±0.3 and 5.3±0.3 mmol/L in the IDDM patients with normoalbuminuria and macroalbuminuria, respectively, and 5.3±0.1 mmol/L in the normal subjects (P=NS) immediately before the endothelial function test. Serum-free insulin concentrations averaged 41±6, 55±9, and 44±5 pmol/L, respectively (P=NS). A 27-gauge unmounted steel cannula (Coopers Needle Works) connected to an epidural catheter (Portex, Hythe) was inserted into the left brachial artery. Drugs were infused at a constant rate of 1 mL/min with infusion pumps (Braun AG and Harvard Apparatus model 22). Subjects rested supine in a quiet environment for 30 minutes after needle placement before any blood flow measurements were made. Normal saline was first infused for 12 minutes. Drugs were then infused in the following sequence: SNP (Roche) 3 and 10 µg/min, ACh (Iolab Corp) 7.5 and 15 µg/min, and L-NMMA (Clinalfa) 4 µmol/min. Each dose was infused for 6 minutes, and the infusion of each drug was separated by infusion of normal saline for 18 minutes, during which blood flow was allowed to return to baseline values. Forearm blood flow was recorded for 10 seconds at 15-second intervals during the last 3 minutes of each drug and saline infusion period. The measurement was performed simultaneously in the infused (experimental) and control arms. Blood flow was recorded with mercury-in-rubber strain-gauge venous occlusion plethysmography (EC 4 Strain Gauge Plethysmograph, Hokanson) combined with a rapid cuff inflator (E 20, Hokanson) and an analog-to-digital converter (McLab/4e, AD Instruments Pty Ltd) connected to a personal computer, as previously described.23 The mean of the final five measurements of each recording period was used for analysis.

Reflex Forearm Blood Flow Response to Cold Immersion
To assess thermoregulatory reflex sympathetic vasomotor function, we measured the forearm blood flow response to a standardized cold stimulus.17 This measurement was performed immediately after normoglycemia was achieved and before insertion of the intrabrachial cannula (vide supra). The subjects were supine during the test. The right forearm was immersed into a container containing iced water for 30 seconds. Forearm blood flow was measured in the left forearm before cold immersion and immediately after the 30-second cold immersion period for a total of 5 minutes by use of venous occlusion plethysmography as described above.

Cardiovascular Autonomic Function Tests
The subjects underwent autonomic cardiovascular nervous function tests24 25 in the following order: controlled and deep breathing test, spectral analysis of heart rate variability, Valsalva test, isometric hand grip test, and orthostatic test. In the controlled breathing test, a measure of both parasympathetic and sympathetic inputs into heart rate control,24 a quiet sound signal was given to pace the inspirium and expirium for 2 seconds each. This pattern was maintained for 5 minutes, during which the RR intervals were measured from the ECG. The RMSSD was calculated. The frequency domain analysis of heart rate variability was done by use of spectral analysis of RR interval variability with the CAFTS system (Medicro Oy). After the RR interval signal was detrended, a least-mean-squares autoregressive model with a model order of 14 was used to obtain the power spectral estimate of RR interval variability. TP was determined in the frequency range of 0 Hz to 0.5 times heart rate in hertz. LF was determined in the frequency range of 0.04 to 0.15 Hz, and it is thought to be mediated by both parasympathetic and sympathetic pathways.26 HF was determined in the frequency range of 0.15 to 0.40 Hz, and it is thought to be mediated by parasympathetic pathways.26 The signal powers were calculated as integrals under the respective part of the power spectral density function and were expressed in absolute units (milliseconds squared) and as a ratio (LF/HF) as a measure of "sympathovagal balance."27 In the deep breathing test, a test of vagal heart rate control,24 the duration of inspirium and expirium was 5 seconds for both for 40 seconds (four respiratory cycles). The ratio of the longest and shortest RR intervals was determined from the ECG for each respiratory cycle, and the mean of the four ratios was taken as the E/I ratio. In the Valsalva test, a measure of both parasympathetic and sympathetic function,24 the subjects blew into a manometer, thereby maintaining an intrathoracic pressure of 40 mm Hg for 15 seconds. The ratio of the shortest RR interval during the expiratory strain and the longest RR interval during the 20 seconds after the end of the strain was calculated (Valsalva ratio). In the isometric hand grip test, the subjects squeezed a dynamometer in their dominant hand for 3 minutes at a force corresponding to 30% of their maximal squeezing force. Heart rate and blood pressure were measured at rest before the test and at the end of the hand grip. In the orthostatic test, the subjects actively stood up after resting quietly supine for 5 minutes. Heart rate and blood pressure were measured at rest and 1, 3, 5, and 7 minutes after the subjects stood up.

To determine the possible effect of hyperglycemia on variation in autonomic function tests, all tests were performed twice in seven patients with IDDM (age, 28 to 38 years; body mass index, 21.0 to 33.2 kg/m2; duration of diabetes, 20 to 28 years) during hyperglycemic (plasma glucose, 12.7±0.6 mmol/L) and normoglycemic (glucose, 5.9±0.2 mmol/L) conditions. These studies were performed by either increasing plasma glucose concentrations by a meal containing approximately 300 to 400 kcal or by lowering glucose by a low-dose intravenous insulin infusion, as described by Mokan and Gerich.28 Serum-free insulin concentrations were comparable during the tests performed under hyperglycemic (70±10 pmol/L) and normoglycemic (95±26 pmol/L) conditions. The results of the autonomic function tests performed under hyperglycemic and normoglycemic conditions were as follows: E/I ratio, 1.3±0.04 and 1.3±0.1 (P=NS); Valsalva ratio, 1.6±0.1 and 1.7±0.1 (P=NS); increase in diastolic blood pressure in hand grip test, 17±2 and 15±3 mm Hg (P=NS); decrease in systolic blood pressure in orthostatic test (7 minutes), -4.4±5.6 and -8.4±5.5 mm Hg (P=NS); RMSSD, 14±3 and 14±3 ms (P=NS); LF, 308±105 and 335±84 ms2 (P=NS); HF, 112±34 and 101±38 ms2 (P=NS); LF/HF, 6.0±1.8 and 6.7±1.6 (P=NS); and TP, 744±204 and 818±211 ms2 (P=NS). The intraindividual coefficients of variation were as follows: E/I ratio, 3.7%; Valsalva ratio, 4.7%; hand grip test, 14%; orthostatic test, 25%; RMSSD, 16%; LF, 25%; HF, 34%; LF/HF, 19%; and TP, 18%. These coefficients of variation are lower than those previously reported for nondiabetic subjects29 and can be classified as very good for all except the HF test according the French Group for Research and Study of Diabetic Neuropathy.30

Other Measurements
Plasma glucose concentrations were measured in duplicate with the glucose oxidase method by use of the Beckman Glucose Analyzer II (Beckman Instruments). HbA1c was measured by high-performance liquid chromatography with the fully automated Glycosylated Hemoglobin Analyzer System (BioRad). Urine albumin was measured by the immunoturbidimetric (Hitachi Ltd) method with an antiserum against human albumin (Orion Diagnostica). Fat-free mass was measured by a single frequency bioelectrical impedance device (Bio-Electrical Impedance Analyzer System, model BIA-101A).

Statistical Analysis
Data between the three study groups were analyzed by use of ANOVA followed by a pairwise comparison with Fisher's least significant difference test. Simple correlations between selected study variables were calculated by use of Spearman's rank correlation coefficient. Multiple linear regression analysis was used to analyze the causes of variation in parameters of endothelial function and autonomic neuropathy. UAER was log transformed to normalize its distribution for multiple linear regression analysis. All calculations were made with the SYSTAT statistical package (SYSTAT Inc). All data are expressed as mean±SEM.


*    Results
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 up arrowMethods
 *Results
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Blood Flow Responses to ACh, SNP, and L-NMMA
Basal blood flow in the experimental arm before drug infusions averaged 2.3±0.2 and 2.8±0.2 mL·dL-1·min-1 in the IDDM patients with normoalbuminuria and macroalbuminuria, respectively (P=NS), and 2.6±0.2 mL·dL-1·min-1 in the normal subjects (P=NS versus both IDDM groups). Patients with IDDM with macroalbuminuria exhibited hyperresponsiveness to both doses of ACh in the experimental arm compared with the patients with normoalbuminuria and the normal subjects (Fig 1Down). During the low-dose ACh infusion, blood flow averaged 7.5±1.0, 6.1±0.9, and 10.5±1.0 mL·dL-1·min-1 in normal subjects and IDDM patients with normoalbuminuria and macroalbuminuria, respectively (P<.01 for blood flow in the macroalbuminuric IDDM group versus other groups; P<.05 for macroalbuminuric versus normoalbuminuric IDDM patients). Blood flow during high-dose ACh averaged 8.6±1.2, 7.9±1.2, and 12.3±1.3 mL·dL-1·min-1, respectively (P<.01 for IDDM patients with macroalbuminuria versus other groups; P<.05 for macroalbuminuric versus normoalbuminuric patients). The blood flow response to low-dose SNP was also significantly higher in the macroalbuminuric IDDM patients than in the other two groups (Fig 1Down). Within the patients with IDDM, blood flow during the low (r=.49, P<.02) and high (r=.74, P<.001) doses of ACh and SNP were significantly correlated. During infusion of L-NMMA, blood flow was not significantly different between the groups (2.0±0.3, 2.4±0.2, and 2.4±0.3 mL·dL-1·min-1 in normal subjects and IDDM patients with normoalbuminuria and macroalbuminuria, respectively). Forearm blood flow during the vasoactive drug and saline infusions in the control arm was stable and comparable between the groups (data not shown).



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  		Figure 1. Forearm blood flows in the experimental arm in the normal subjects (Cont, n=11) and IDDM patients with normoalbuminuria (Normo, n=12) and macroalbuminuria (Macro, n=10) during intrabrachial infusion of ACh (7.5 and 15 µg/min; top) and SNP (3 and 10 µg/min; bottom). Data are shown as mean±SE. *P<.05 for Macro vs Cont subjects; ++P<.02 for Macro vs Normo subjects.

Blood Flow Response to Cold Immersion and Other Tests of Autonomic Nervous Function
The decrease in blood flow in response to cold immersion was severely impaired in the IDDM patients with macroalbuminuria, because blood flow decreased by only 19±6% in this group compared with 54±7% in the normal subjects and 39±5% in normoalbuminuric IDDM patients (P<.001 for macroalbuminuric IDDM patients versus normal subjects; P<.05 versus normoalbuminuric patients; Fig 2Down). Absolute flow during cold immersion in the contralateral arm was almost twofold higher in the IDDM patients with macroalbuminuria (2.1±0.2 mL·dL-1·min-1) than in those with normoalbuminuria (1.3±0.2 mL·dL-1·min-1) or the normal subjects (1.1±0.2 mL·dL-1·min-1; P<.01 for macroalbuminuric IDDM patients versus normal subjects; P<.05 versus normoalbuminuric patients).



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  		Figure 2. Blood flow during cold immersion (top) and the percent decrease in forearm blood flow compared with baseline (bottom). **P<.01, ***P<.001 for IDDM patients with macroalbuminuric (Macro) vs normal (Cont) subjects; +P<.05 for Macro vs normoalbuminuric (Normo) subjects.

The macroalbuminuric patients with IDDM had evidence of autonomic dysfunction during controlled breathing and during the Valsalva maneuver compared with normal subjects (Table 2Down). The IDDM patients with normoalbuminuria exhibited significant impairments in autonomic function during the Valsalva maneuver and controlled breathing (Table 2Down).


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  		Table 2. Results of Autonomic Nervous System Tests in the Study Groups

Determinants of the Blood Flow Response to ACh and SNP
To search for the correlates of the variation in the blood flow responses to endothelium-independent and endothelium-dependent vasoactive responses, we calculated the correlation coefficients between parameters such as body mass index, age, HbA1c, duration of diabetes, UAER, serum cholesterol, creatinine, blood pressure, and blood flow responses to ACh, SNP, and L-NMMA. Within the IDDM patients, none of these parameters correlated with forearm blood flow during the vasoactive drug infusions (data not shown). There was, however, a significant inverse correlation between several measures of autonomic function and vascular hyperresponsiveness to ACh and SNP within the group of IDDM patients. For example, the heart rate response to the Valsalva maneuver, a measure of sympathetic and parasympathetic autonomic function,31 was closely inversely related both to ACh (Fig 3Down) and SNP (Fig 4Down) responses. A significant inverse correlation between the blood flow response to low-dose ACh and the Valsalva ratio was also observed within the IDDM patients with macroalbuminuria (r=-.75, P<.01) and normal subjects (r=-.83, P<.01) but not the patients with normoalbuminuria (r=-.49, P=NS). The respective correlation coefficients for the high-dose ACh were -0.85 (P<.001), -0.71 (P<.05), and -0.53 (P=NS); for the low-dose SNP, -0.95 (P<.001), -0.46 (P=NS), and -0.48 (P=NS); and for the high-dose SNP, -0.85 (P<.01), -0.25 (P=NS), and -0.54 (P=NS). Significant inverse relationships were also observed between several tests that predominantly reflect function of the sympathetic nervous system and the blood flow responses to SNP and ACh (Table 3Down).



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  		Figure 3. Relationship between the Valsalva ratio and forearm blood flow during intrabrachial infusion of ACh at rates of 7.5 (top) and 15 (bottom) µg/min in patients with IDDM.



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  		Figure 4. Relationship between the Valsalva ratio and forearm blood flow during intrabrachial infusion of SNP at rates of 3 (top) and 10 (bottom) µg/min in patients with IDDM.


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  		Table 3. Simple Correlations Between Forearm Blood Flow Responses to Intrabrachially Administered Vasoactive Drugs and Autonomic Function Tests Thought to Predominantly Reflect Function of the Sympathetic Nervous System in Patients With IDDM

The association between autonomic function and endothelium-independent and endothelium-dependent vasodilation was confirmed by use of multiple linear regression analysis. For example, the Valsalva ratio (P<.02) but not UAER was an independent determinant of blood flow during infusion of the low (R2=63%, P<.01) and high (R2=68%, P<.01) doses of SNP. The Valsalva ratio (P<.02) but not UAER was also an independent determinant of blood flow during infusion of the low (R2=69%, P<.05) and high (R2=65%, P<.02) doses of ACh. The Valsalva ratio (P<.05 and P<.01 for low and high SNP doses, respectively; P<.05 for both low and high ACh doses) but not the use of ACE inhibitors was an independent determinant of the blood flow response in ANOVA comparisons of patients using (n=6) and not using (n=12) ACE inhibitors with the Valsalva ratio as the covariate.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Previous studies addressing endothelial function in patients with IDDM have yielded conflicting data. The blood flow response to SNP has been either decreased12 or normal.11 13 14 15 16 Similarly, the blood flow response to ACh, metacholine, or carbachol has been decreased15 16 or, more frequently, normal.11 12 13 14 Sex confounds comparison of forearm vascular responsiveness because of the influence of forearm length on inactivation of ACh,32 but decreased12 and normal14 blood flow responses to SNP and normal responses to ACh have been found even within male IDDM patients. Other factors that might have contributed to the heterogeneous results include the ambient glucose concentration in the diabetic patients during the endothelial function tests, long-term glycemic control, and the presence of normoalbuminuria or microalbuminuria, but even after these parameters are taken into consideration, no consistent picture emerges from these studies.

In none of the above studies addressing endothelial function in IDDM have possible abnormalities in neural control of blood flow responses been quantified. In the present study, we found striking associations between autonomic function and the response of forearm resistance vessels to both SNP and ACh. In the cold immersion test, reflex vasoconstriction was blunted in IDDM patients with macroalbuminuria (Fig 2Up). This abnormality, as well as the correlation between the several measures of autonomic sympathetic innervation and basal and SNP- and ACh-stimulated blood flows (Table 3Up), are consistent with the presence of sympathetic denervation in our study subjects. The weaker correlation between the cold immersion test result and SNP- and ACh-stimulated blood flows compared with other autonomic function tests (Table 3Up) may be explained by the fact that the cold immersion test could also be influenced by the integrity of small myelinated and unmyelinated sensory fibers involved in exteroception.33 Together, these data suggest that the paradoxical hyperresponsiveness of forearm blood flow to SNP and ACh could be explained by failure of the sympathetic autonomic nervous system to counteract vasodilation induced by exogenously administered vasodilators. ACh acts as a vasodilator through both stimulation of endogenous NO release from endothelial cells and inhibition of adrenergic neurotransmission.34 The hyperresponsiveness to this agent is therefore unlikely to reflect exaggerated endogenous NO release or hypersensitivity to NO35 but might be due to hypersensitivity of muscarinic receptors on adrenergic nerves or endothelial cells.36 The latter interpretation would be consistent with the normal decrease in blood flow by L-NMMA in patients with macroalbuminuria and most severe autonomic dysfunctions. Regarding the explanation for the exaggerated blood flow response to SNP, there is again no reason to assume that hypersensitivity to NO, as described by Moncada et al,35 was responsible because the blood flows were comparable between the groups during infusion of L-NMMA. A direct antagonistic effect of SNP on vasoconstriction mediated by the sympathetic nervous system also is unlikely because autonomic dysfunction would impair rather than strengthen such an effect. It therefore seems more likely that the increased response to SNP in IDDM patients with autonomic nervous system dysfunction reflected a failure to counteract a small systemic depressor effect of SNP through the baroreceptor reflex that mediates reflex sympathetic vasoconstriction. Because it was not technically possible to follow changes in blood pressure during the endothelial function test, this possibility remains speculative at present.

The present data may help to clarify some previous observations regarding endothelial function in IDDM and the apparent normality of the vascular responses of the normoalbuminuric group in the present study. Although studies with isolated vessels have convincingly demonstrated the ability of acute and chronic hyperglycemia to blunt endothelium-dependent vasodilation, this parameter has been normal in four11 12 13 14 of the six studies performed in IDDM patients with normoalbuminuria and microalbuminuria. In the present study, marked abnormalities in autonomic function were associated with hyperresponsiveness to nitrovasodilators, whereas the responses were normal in equally hyperglycemic patients with less severe abnormalities in autonomic function. The latter data are in keeping with previous data showing predominantly normal responses to both ACh11 12 13 14 and SNP.11 13 14 15 16 This normality may, however, be more apparent than real if hyperglycemia-induced defects in vasodilation2 3 4 5 are counteracted by hyperresponsiveness to nitrovasodilators owing to altered neurovascular control. This is also why the relationships between vascular responses to ACh and SNP and indexes of autonomic function were analyzed within subgroups of IDDM patients and normal subjects rather than by pooling all data together.

Although the risk of premature cardiovascular death is extremely high in IDDM patients with diabetic nephropathy,7 the exact nature of the cardiovascular disease and its causes are far from clear. Traditional risk factors such as an elevated cholesterol concentration, blood pressure, or cigarette smoking explain only a small part of the excessive cardiovascular mortality of patients with diabetic nephropathy.7 Poor glycemic control increases the prevalence and progression of diabetic nephropathy and neuropathy.37 Chronic hyperglycemia also appears to predispose patients with NIDDM to develop coronary artery disease, but whether this is also true for patients with IDDM is currently uncertain. Autonomic neuropathy may also contribute to the increased cardiovascular mortality in IDDM. Its presence is associated with an increased prevalence of silent ischemia,8 9 QT-interval lengthening,9 cardiorespiratory arrest,8 9 poor outcome of myocardial infarction,38 and increased mortality.8 9 The present study shows that patients whose autonomic control of heart rate variation is impaired also have abnormal blood flow responses to cold and to ACh and SNP in their forearm resistance vessels. Because endothelial dysfunction in forearm resistance vessels1 seems to be associated with similar abnormalities in the coronary arteries, it is possible that regulation of blood flow by autonomic or other stimuli is abnormal not only in forearm resistance vessels but also in the heart. Thus, autonomic dysfunction may not be merely a confounding variable in the interpretation of endothelial function tests in patients with IDDM; it may also be of clinical significance in the pathogenesis of cardiovascular complications in these patients. In this context, it is of interest that the long-term prognosis after myocardial infarction in nondiabetic subjects is linked to heart rate variability.39


*    Selected Abbreviations and Acronyms
 
ACh = acetylcholine
E/I = expiration-to-inspiration ratio
HF = high-frequency power
IDDM = insulin-dependent diabetes mellitus
L-NMMA = L-N-monomethyl-arginine
LF = low-frequency power
NO = nitric oxide
RMSSD = square root of the mean of the square of successive RR-interval differences
SNP = sodium nitroprusside
TP = total power
UAER = urinary albumin excretion rate


*    Acknowledgments
 
This work was supported by grants from the Academy of Finland, the Sigrid Juselius, and Ahokas (all for Dr Yki-Jarvinen) foundations. We thank Sari Hamalainen for excellent technical assistance, Soile Aarnio for drawing the figures, and the volunteers for their help. We also greatly appreciate the help of John Cockcroft, MD, Department of Medicine, Division of Therapeutics, University Hospital, Queen's Medical Centre, Nottingham, Great Britain, for fruitful collaboration.

Received June 25, 1996; revision received October 2, 1996; accepted October 13, 1996.


*    References
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 up arrowIntroduction
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 *References
 

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