the study group consisted of 48 patients with type 1 diabetes ( 26 women and 22 men ; age , 39.8  6.3 years ) recruited from the regional diabetological centre of the medical university of gdansk and 24 volunteers who were free of any chronic diseases ( 13 women and 11 men ; age , 38.0  6.7 years ) ( see table 2 ) . we included patients with a minimum diabetes duration of 4 years and who were free of focal neurologic deficits and renal insufficiency . additional exclusion criteria were pregnancy and pathological obesity ( bmi > 35 kg / m ) . all examinations were performed at the same time of day ( between 8:00 a.m. and 1:00 p.m. ) . all study subjects were asked to stop drinking coffee and to stop smoking tobacco on examination day and to avoid sleep deprivation on previous days . the study protocol included medical history , neurologic examination , fundoscopy , and laboratory testing . the study protocol was approved by the medical ethics committee of the medical university of gdansk ( nkebn/335/2008 , nkebn/33560/2009 , nkebn/204/2010 ) . on entry to the study , patient histories were obtained , including information on past and current disorders as well as comorbid conditions and cigarette smoking . obesity was defined as bmi 30 kg / m , and overweight was defined as bmi in the range of 2530 kg / m . diabetic neuropathy was diagnosed using the criteria defined by the neurologic symptoms score and neurologic disability score based on patient symptoms and neuropathic deficits found on neurologic examination ( 20 ) . laboratory examinations in patients with type 1 diabetes included microalbuminuria , albuminuria / creatinine index , total serum cholesterol , ldl cholesterol , hdl cholesterol , triglycerides , and glycated hemoglobin ( hba1c ) . hyperlipidemia was diagnosed if total cholesterol was > 175 mg / dl and/or triglycerides were > 150 mg / dl and/or ldl cholesterol was > 100 mg / dl . hyperlipidemia also was diagnosed if hdl level was < 40 mg / dl in males or < 50 mg / dl in women , or if cholesterol - lowering / triglyceride - lowering medications were used ( 21 ) . in the control group , hyperlipidemia was diagnosed if total cholesterol and/or triglycerides and/or ldl cholesterol exceeded 190 , 150 , and 130 mg / dl , respectively , or if hdl level was < 40 mg / dl in males or < 50 mg / dl in women ( 22 ) . based on the criteria of the european association for the study of diabetes , microalbuminuria was defined as the albuminuria / creatinine index in a random spot collection ( > 30 g / mg creatinine ) or overnight collection ( > 20 g / min ) in two out of three urine collections repeated at intervals of up to 6 months . the european association for the study of diabetes recommendations corresponded to protein excretion > 300 mg/24 h ( 21 ) . retinopathy was recognized on fundoscopy performed by an experienced ophthalmologist certified by the polish ophthalmic society . retinopathy was graded according to the stages of diabetic retinopathy defined by the american academy of ophthalmology ( 23 ) . subjects participating in the study were asked not to dispatch the skin of their fingernails at least for 2 weeks before the start of the study . during examination , patients remained in a comfortable sitting position with the hand conveniently supported and laid stably under the capillaroscope . tags were drawn on their nail folds to establish areas for further examination during repeated measurements . the room temperature was controlled by air conditioning and was kept the same during all tests . body temperature was controlled with the use of a contactless thermometer and was within normal range in all examined patients and controls . the nail - fold capillaries were made transparent by adding a drop of immersion oil , and images were generated by a lens providing a magnification of 200. images were taken using a digital camera ( 5mpx ; opta - tech , warsaw , poland ) attached to the capillaroscope ( opta - tech , cs - creative solutions group , warsaw , poland ) and archived on disc . the software for image analysis was written in c++ language according to concepts formulated by the authors ( 24 ) . offline computer analysis included the following steps : rotation of the image ; selection of the part of image for further analysis ; tuning process of selected area ; recognition of capillaries ; and final acceptation of achieved results . the images with less than five capillaries in each row detected were not considered for statistical analysis . the image analysis allows determination of the mean distance between successive capillaries ( distance ) and the ratio between area of the capillaries and total area of determined rows ( coverage ) . the capillaroscopy was performed twice , before and after 30 min of l - arginine infusion ( 30 g 21% [ wt / vol ] l - arginine hydrochloride solution [ b. braun melsungen ag , germany ] in 100 ml sodium chloride ) . the l - arginine infusion was performed intravenously within 30 min into intermediate cubital vein of the arm ipsilateral to the capillaroscopy . the skin microcirculation reactivity was expressed as the difference between the values of distance ( distance ) and coverage ( coverage ) obtained before and after infusion of l - arginine . two - hundred fifty - two random coded images were used for the reproducibility study . the unbiased intraclass correlation coefficient between the sets of data obtained was considered to be an acceptable measure of agreement if intraclass correlation coefficient ( mean and 90% confidence interval ) for distance and coverage yielded 0.64 ( 0.580.70 ) and 0.63 ( 0.560.70 ) , respectively . differences between groups were analyzed with the student ttest in the case of normally distributed variables ( age ) or with mann - whitney u test in the case of non - normally distributed variables ( hba1c , parameters describing the skin microcirculation ) . the unbiased intraclass correlation coefficient was considered as an acceptable measure of agreement between the sets of repeated data . median age of onset of diabetes coincided with the time of puberty , and therefore was used as the threshold value to divide the whole diabetic group into subgroups ( table 1 ) . subgroup analysis in patients with type 1 diabetes the 2 test was used to compare the proportion of genders and the presence of smoking habit , obesity or overweight , hypertension , hyperlipidemia , microalbuminuria , physical activity , contraception , microangiopathy , and treatment with statins , -blockers , angiotensin receptor blockers , or ace inhibitors . patient histories were obtained , including information on past and current disorders as well as comorbid conditions and cigarette smoking . obesity was defined as bmi 30 kg / m , and overweight was defined as bmi in the range of 2530 kg / m . diabetic neuropathy was diagnosed using the criteria defined by the neurologic symptoms score and neurologic disability score based on patient symptoms and neuropathic deficits found on neurologic examination ( 20 ) . laboratory examinations in patients with type 1 diabetes included microalbuminuria , albuminuria / creatinine index , total serum cholesterol , ldl cholesterol , hdl cholesterol , triglycerides , and glycated hemoglobin ( hba1c ) . hyperlipidemia was diagnosed if total cholesterol was > 175 mg / dl and/or triglycerides were > 150 mg / dl and/or ldl cholesterol was > 100 mg / dl . hyperlipidemia also was diagnosed if hdl level was < 40 mg / dl in males or < 50 mg / dl in women , or if cholesterol - lowering / triglyceride - lowering medications were used ( 21 ) . in the control group , hyperlipidemia was diagnosed if total cholesterol and/or triglycerides and/or ldl cholesterol exceeded 190 , 150 , and 130 mg / dl , respectively , or if hdl level was < 40 mg / dl in males or < 50 mg / dl in women ( 22 ) . based on the criteria of the european association for the study of diabetes , microalbuminuria was defined as the albuminuria / creatinine index in a random spot collection ( > 30 g / mg creatinine ) or overnight collection ( > 20 g / min ) in two out of three urine collections repeated at intervals of up to 6 months . the european association for the study of diabetes recommendations corresponded to protein excretion > 300 mg/24 h ( 21 ) . retinopathy was recognized on fundoscopy performed by an experienced ophthalmologist certified by the polish ophthalmic society . retinopathy was graded according to the stages of diabetic retinopathy defined by the american academy of ophthalmology ( 23 ) . subjects participating in the study were asked not to dispatch the skin of their fingernails at least for 2 weeks before the start of the study . during examination , patients remained in a comfortable sitting position with the hand conveniently supported and laid stably under the capillaroscope . tags were drawn on their nail folds to establish areas for further examination during repeated measurements . the room temperature was controlled by air conditioning and was kept the same during all tests . body temperature was controlled with the use of a contactless thermometer and was within normal range in all examined patients and controls . the nail - fold capillaries were made transparent by adding a drop of immersion oil , and images were generated by a lens providing a magnification of 200. images were taken using a digital camera ( 5mpx ; opta - tech , warsaw , poland ) attached to the capillaroscope ( opta - tech , cs - creative solutions group , warsaw , poland ) and archived on disc . the software for image analysis was written in c++ language according to concepts formulated by the authors ( 24 ) . offline computer analysis included the following steps : rotation of the image ; selection of the part of image for further analysis ; tuning process of selected area ; recognition of capillaries ; and final acceptation of achieved results . the images with less than five capillaries in each row detected were not considered for statistical analysis . the image analysis allows determination of the mean distance between successive capillaries ( distance ) and the ratio between area of the capillaries and total area of determined rows ( coverage ) . the capillaroscopy was performed twice , before and after 30 min of l - arginine infusion ( 30 g 21% [ wt / vol ] l - arginine hydrochloride solution [ b. braun melsungen ag , germany ] in 100 ml sodium chloride ) . the l - arginine infusion was performed intravenously within 30 min into intermediate cubital vein of the arm ipsilateral to the capillaroscopy . the skin microcirculation reactivity was expressed as the difference between the values of distance ( distance ) and coverage ( coverage ) obtained before and after infusion of l - arginine . two - hundred fifty - two random coded images were used for the reproducibility study . the unbiased intraclass correlation coefficient between the sets of data obtained was considered to be an acceptable measure of agreement if intraclass correlation coefficient ( mean and 90% confidence interval ) for distance and coverage yielded 0.64 ( 0.580.70 ) and 0.63 ( 0.560.70 ) , respectively . differences between groups were analyzed with the student ttest in the case of normally distributed variables ( age ) or with mann - whitney u test in the case of non - normally distributed variables ( hba1c , parameters describing the skin microcirculation ) . the unbiased intraclass correlation coefficient was considered as an acceptable measure of agreement between the sets of repeated data . median age of onset of diabetes coincided with the time of puberty , and therefore was used as the threshold value to divide the whole diabetic group into subgroups ( table 1 ) . subgroup analysis in patients with type 1 diabetes the 2 test was used to compare the proportion of genders and the presence of smoking habit , obesity or overweight , hypertension , hyperlipidemia , microalbuminuria , physical activity , contraception , microangiopathy , and treatment with statins , -blockers , angiotensin receptor blockers , or ace inhibitors . all patients and volunteers were free of diabetic foot , orthostatic hypotension , and chronic renal insufficiency , as determined by physical examination and biochemical analysis . there were no significant differences regarding age , sex , cigarette smoking , bmi and obesity , hormonal contraceptive usage , or regular physical activity between patients and control subjects . the percentage of subjects with dyslipidemia was significantly higher in type 1 diabetic patients than in controls ( table 2 ) . baseline characteristics and the comparison of the skin capillary data between patients with type 1 diabetes and control subjects the coverage was significantly higher and the distance was significantly lower in patients with type 1 diabetes than in control subjects before l - arginine infusion ; the differences between groups regarding both coverage and distance diminished after l - arginine infusion ( table 1 ) . the duration of the disease and patient age did not correlate with distance and coverage . similarly , there were no significant correlations between age and distance and coverage in the controls . in the whole studied cohort ( n = 72 ) , regardless of the group , there were significant correlations between age and both distance and coverage ( r = 0.27 , p = 0.02 and r = 0.025 , p = 0.04 , respectively ) . we found no significant differences in coverage and distance in subgroups of patients with type 1 diabetes distinguished by the presence of diabetic retinopathy , neuropathy , overt nephropathy , microalbuminuria , hypertension , or hyperlipidemia . additionally , no differences in coverage and distance were found between groups of patients distinguished on the basis of treatment with ace inhibitors , angiotensin receptor blockers , or -blockers , or hba1c level equal to 7% . within the group of patients with type 1 diabetes , the onset of diabetes correlated positively with distance ( r = 0.43 ; p = 0.002 ) and negatively with coverage ( r = 0.39 ; p = 0.006 ) . when subgroups were created according to the average age of onset of diabetes ( 19.7 years ; median , 19 years ) , further analysis showed a significantly lower coverage and significantly higher distance in patients with later onset of type 1 diabetes ( group b ) in comparison with those with earlier onset of type 1 diabetes ( group a ) ( table 2 , right panel ) . patients with later onset of type 1 diabetes were characterized by similar bmi , shorter diabetes duration ( p = 0.002 ) , and older age ( p = 0.04 ) , as well as higher triglyceride ( p = 0.03 ) and urine creatinine levels ( p = 0.01 ) than in those with earlier onset . neither subgroup of the type 1 diabetes group differed from the control groups according to age . the analysis of obtained data showed no correlation between the duration of diabetes and microvascular parameters in either of the subgroups . the group with earlier onset of diabetes had significantly greater skin microcirculation reactivity described by coverage and distance ( p = 0.02 ; fig . 1 ) and a higher incidence of retinopathy ( 64% for group a vs. 26% for group b ; p = 0.02 ) , but no neuropathy , nephropathy , or any of these microangiopathies in comparison with the diabetes group with later onset of the disease . both variables were significantly different between the group with later but not with earlier onset of diabetes in comparison with the control group ( fig . 1 ) . comparison of changes in skin microvascular reactivity parameters attributable to l - arginine infusion between studied groups . the correlation analysis between distance and coverage with the duration of the disease in subgroups with earlier and later onset of diabetes did not reach statistical significance . the influence of l - arginine on no - dependent vasodilatation has been the subject of several studies regarding diabetes ( 2528 ) . however , the regional differences in the vascular response to l - arginine are not fully determined . to our best knowledge , the relationship between microangiopathy and the skin microcirculation reactivity , as revealed by capillaroscopy , in response to l - arginine infusion in long - duration type 1 diabetes has not yet been described in the literature . our results showed a lack of capillary response to l - arginine in patients with type 1 diabetes in contrast to control subjects when an effect was observed . the l - arginine - related coverage in the control group was significantly greater than zero ( p = 0.02 ) , whereas for patients with type 1 diabetes there was no change . ( 12 ) , who demonstrated maximal rest recruitment of the skin microcirculation in response to postocclusive hyperemia in diabetic patients . it is noteworthy that similar conclusions were obtained , even though there were essential differences in the mechanism of action between the two testing methods . the l - arginine test describes endothelial reactivity dependent on no action solely , whereas the main mechanisms involved in the ischemic test are shear stress and its consequences ( 29 ) . in studied patients with type 1 diabetes , the skin microcirculation after l - arginine infusion did not differ between patients with and without microangiopathy . the skin microcirculation reactivity also was independent of the metabolic state described by glycemia and hba1c level or hyperlipidemia . the analysis revealed a significantly negative correlation between the microcirculation reactivity and the onset of diabetes ( r = 0.39 ; p < 0.05 ) , whereas no correlation with the duration of disease was found . the average onset of disease in study subjects was 19.7 years ( median , 19 years ) . the group with an age of onset of disease after 19.7 years ( group b , later onset of type 1 diabetes ) was characterized by lower skin microreactivity despite shorter duration of disease . this finding was in contrast to that of patients with an onset of disease before the age of 19.7 years ( group a , earlier onset of type 1 diabetes ) in which a higher prevalence of retinopathy was observed . moreover , our results showed no significant differences between controls and group a regarding skin microcirculation parameters . the same comparison for group b showed substantially lower significance levels than for all cohorts of patients with diabetes . the association between the prevalence of retinopathy and the onset of diabetes before puberty is well known . ( 30 ) reported that prepubertal diabetes duration is related to the prevalence of retinopathy in adult patients . this conclusion is consistent with our results , which show significantly more retinopathy in the group with the earlier onset of type 1 diabetes . however , the relationship between the nail - fold microcirculation and microangiopathy in type 1 diabetes is still a matter of debate in the literature . meyer et al . ( 9 ) demonstrated a correlation between the presence of diabetic microangiopathy and pathological capillaroscopic images . ( 31 ) did not observe effects of the presence of diabetic neuropathy on capillary blood flow increase attributable to neurovascular stimulation . our conclusions are in contrast to others who have pointed out that endothelial dysfunction is an important predictor of microangiopathic complications ( 1,32,33 ) . this could be partially explained by the fact that these studies were performed in younger cohorts with a shorter duration of diabetes ( 12 ) and used the postocclusive reactive hyperemia method or venous congestion to achieve maximal recruitment of capillaries ( 5,10 ) . moreover , in our study we used a quantitative approach in contrast to qualitative assessment of nail - fold circulation as the cited authors did . in our study group , the reactivity of the skin microvasculature did not correlate significantly with bmi as was found in the study by tibiri et al . it is noteworthy that the patients with pathological obesity ( bmi > 35 kg / m ) were excluded by protocol . the limitation of our study is the fact that hyperlipidemia was present in 42% of the subjects in the healthy group . this is two - times the estimated percentages for the polish low global risk population , which are 19% and 27% for men and women , respectively ( 34 ) . it should be also noted that because of the relatively small number of studied subjects , our study was not able to confirm unambiguously the findings already established in the literature , such as the association between skin microcirculation parameters and the subgroup parameters ( e.g. , duration of diabetes and hba1c ) . we used l - arginine infusion as the selective stimulator of endothelial vasodilatory function of skin microcirculation . we observed a physiological response to l - arginine infusion in the control group and no response in the diabetic group . these data reflect the impairment of reactivity of skin microcirculation in response to l - arginine in type 1 diabetes . the response to l - arginine differed in subgroups of type 1 diabetes . no coverage and significant decrease in distance suggest paradoxical deterioration after l - arginine infusion in the subgroup with later onset of type 1 diabetes . the lack of significant response after l - arginine infusion regarding coverage and distance in patients with earlier onset of type 1 diabetes , and no difference with the control group allows for the hypothesis that these patients may benefit from supplementation with l - arginine as a substrate for no . the analysis we performed shows that the relationship between skin capillary reactivity and retinopathy presence ( table 1 ) can not be clearly characterized by parameters such as age , duration of the disease , metabolic status , treatment , or presence of concomitant diseases . based on the performed analysis , we can speculate that these two vascular beds may be affected asynchronously and some other factors are in play . further studies of the relationship between skin microcirculation and retinopathy presence need a specifically designed protocol with repeated assessments of both vascular beds . based on our results , we postulate that regular assessment of microvascular reactivity by capillaroscopy may be a useful method for identification of microangiopathic complications in patients with later onset of type 1 diabetes . based on our results , we postulate that regular assessment of skin microvascular reactivity by capillaroscopy and l - arginine infusion may extend the spectrum of methods used in microangiopathy studies in type 1 diabetes .
objectivethe aim of our study was to evaluate the vasodilatory effect of l - arginine infusion on the skin microcirculation and to assess the relationship between this effect and the presence of microangiopathy in patients with type 1 diabetes.research design and methodscapillaroscopy was performed before and after l - arginine infusion in 48 diabetic patients ( 26 women and 22 men ; age , 39.8  6.3 years ) and 24 volunteers free of any chronic disease ( 13 women and 11 men ; age , 38.0  6.7 years ) . 
 the skin microcirculation reactivity , as expressed by the percentage of area covered by capillaries ( coverage ) and the distance between capillaries ( distance ) , and the relationship between microcirculation reactivity and the presence of microangiopathic complications were assessed.resultsthe distance before l - arginine infusion was significantly lower in patients than in controls ( 221 [ 153311 ] vs. 240 [ 185356 ] m ; p = 0.02 ) and did not differ after l - arginine infusion ( 223.5 [ 127318 ] vs. 242.5 [ 181341 ] m ; p = 0.27 ) . 
 the difference between the coverage values obtained before and after l - arginine infusion ( coverage ) was significantly different from zero in the control group but not in the diabetes group . 
 patients with later onset of diabetes were characterized by decreased skin microcirculation reactivity when compared with patients with earlier onset of diabetes ( 1.18 [ 5.07 to 11.60 ] vs. 1.36 [ 6.00 to 8.06 ] ; p = 0.02 ) despite the higher prevalence of retinopathy in patients with earlier onset of diabetes ( 64% vs. 26% ; p = 0.02).conclusionsskin microvascular reactivity is impaired in patients with later onset of type 1 diabetes . 
 capillaroscopy with l - arginine infusion is useful for the identification of skin microangiopathy in type 1 diabetes .