atherosclerosis and its vascular damage are the leading cause of mortality and morbidity in patients with diabetes .
recently , several studies have shown that cardiovascular diseases are the first cause of premature death among individuals with type 1 diabetes , demonstrating that although there have been substantial improvements in survival during the past 50 years , still challenges remain .
although diabetes - related vascular complications are uncommon in the pediatric age , early functional and structural abnormalities may be present just a few years after the onset of type 1 diabetes .
therefore , early diagnostic and therapeutic approaches are needed for youth with type 1 diabetes at risk for a premature vascular disease .
a surrogate marker of cardiovascular disease is endothelial dysfunction , which is the inability of the artery to sufficiently dilate in response to an appropriate endothelial stimulus .
the reactive hyperemia peripheral artery tonometry technique is used for a few years now as a noninvasive test to assess for early vascular changes in high - risk patient groups . in a previous study
, we evaluated the prevalence of endothelial dysfunction measured as mean of reactive hyperemia index ( rhi ) , in a cohort of adolescents with type 1 diabetes .
surprisingly a low rhi score was observed in 76.7% of patients , showing a dramatic prevalence of early endothelial dysfunction among type 1 diabetes individuals , even in the pediatric age .
endothelial cells have got numerous functions crucial for maintaining intravascular homeostasis : they play an important role in vascular tone regulation , hemostasis , and fibrinolysis and in the production of several substances [ 6 , 7 ] . in patients with diabetes , hyperglycemia and related pathological biochemical processes trigger damage to the endothelial cells causing their dysfunction .
the dysfunctional endothelium adopts prothrombotic , proinflammatory , and vasoconstrictive phenotype promoting the early development of atherosclerosis [ 3 , 6 , 7 ] .
alpha - lipoic acid is a potent mitochondrial antioxidant agent that acts by multiple mechanisms promoting anti - inflammatory and antithrombotic pathways and positively influencing the nitric oxide mediated vasodilatation . in most european countries
alpha - lipoic acid is licensed and used as treatment in patients with neuropathic symptoms . moreover ,
many studies suggest the potential role of alpha - lipoic acid in prevention and treatment of atherosclerosis and related cardiovascular disease [ 10 , 11 ] .
this hypothesis is based on similar results obtained either in animal models or in patients with diabetes mellitus , especially type 2 diabetes [ 1214 ] . in streptozotocin - induced rats
, it may be assumed that alpha - lipoic acid treatment can protect against impaired vascular responsiveness . in type 2
diabetes patients , alpha - lipoic acid may influence angiogenesis through an effect on some circulating factors including vegf , bfgf , mcp-1 , and il-10 . therefore , the aim of our study was to investigate the effect of alpha - lipoic acid on endothelial dysfunction in youth with type 1 diabetes . this pilot study is a 6-month , double - blind , randomized controlled trial to test the efficacy of an antioxidant diet plus alpha - lipoic acid versus an antioxidant diet alone in improving endothelial dysfunction in adolescents with type 1 diabetes .
this study was a 6-month , prospective , randomized , double - blind , controlled trial conducted in pediatric patients with type 1 diabetes , performed at the pediatric department of the university of milano .
the trial was performed in accordance with the declaration of helsinki . before any trial - related activities signed informed consent
was obtained from all participants aged 16 years or older and from parents or guardians of participants aged younger than 16 years ( assent was obtained from minors ) .
patients with type 1 diabetes were referred for study participation from the outpatient clinic of the university of milano between january 2013 and december 2013 .
seventy - one patients who had been previously evaluated for the presence of endothelial dysfunction   were enrolled in the trial and completed the 6-month study period .
inclusion criteria were type 1 diabetes ( diabetes onset was defined according to ada criteria ) , more than 1 year from diagnosis or confirmed c - peptide negative , age between 12 and 19 years , insulin requirement more than or equal to 0.5  u / kg / day , blood glucose checks more the 3 times per day , and insulin intensive therapy ongoing .
exclusion criteria were preexisting cardiovascular diseases or inflammatory systemic diseases , hypertension and prehypertension ( bp  90th percentile for age , sex , and height ) , eating disorders , obesity ( defined as bmi  95th percentile per age and sex in the 2000 cdc growth chart ) , celiac disease , inflammatory bowel disease or other significant gastrointestinal conditions , systemic glucocorticoid use ( 1-month cumulative use during last year ) or ongoing treatment with glucocorticoid use , anemia , significant multiple food allergies , uncontrolled hypothyroidism or hyperthyroidism , significant mental illness , and pregnancy .
clinical history and physical examination , nutritional records , and biochemical sample were collected at baseline , after 3 months , and after 6 months ; endothelial dysfunction was evaluated at baseline and after 6 months .
weight was measured unclothed to the nearest 0.1  kg using a calibrated balance scale .
blood pressure was measured using a mercury sphygmomanometer , according to the national high blood pressure education program working group . moreover ,
all patients were in intensive insulin therapy , with either multiple daily injections or insulin pump therapy .
blood sampling was performed in the fasting state at 8  a.m. levels of lipids including triglycerides ( tg ) , total cholesterol ( tc ) , ldl - cholesterol , and hdl - cholesterol were evaluated in all subjects with standard laboratory methods .
blood was drawn for hba1c analysis using a fully automated high - performance liquid chromatography system ( variant ii , bio - rad laboratories , munich , germany ) . at baseline and 3 and 6
months a nutritional visit was performed by the registered dietitian of the pediatric department of the  luigi sacco  hospital .
three - day dietary records with nutrition data were evaluated using dedicated software ( metadieta , meteda , san benedetto del tronto , ap , italy ) .
data on dietary intake , reporting the types and amounts of all food and beverages consumed , with data on insulin doses and algorithms used at home , blood glucose results , and physical activity were collected . during the study period a dietary recall
was collected every month ; moreover a nutritional assessment was performed at 3 and 6 months to check the patients ' compliance to the dietary plan assigned
. moreover , registered dietitian evaluated the body composition using tanita bc-418 , il , usa .
peripheral endothelium - dependent vasodilator capacity was estimated by assessing the rhi by means of the endopat 2000 system ( itamar medical ltd . ,
endothelium - mediated changes in vascular tone after occlusion of the brachial artery are reflecting a downstream hyperemic response , which is a measure for arterial endothelial function .
the rh - pat score is calculated as the ratio of the average pulse wave amplitude during 1  min period starting 60  s after cuff deflation divided by the average pulse wave amplitude of a 210  s preocclusion baseline period .
after the clinical assessment at baseline , each patient was randomly assigned to one of the three double - blind , study arms : 10.000 oxygen radical absorbance capacity units ( orac ) antioxidant diet plus alpha - lipoic acid ( n = 25 , group 1 ) , 10.000 orac antioxidant diet plus placebo ( n = 27 , group 2 ) , and controls ( n = 19 , group 3 ) , with no changes in dietary habits .
supplementation with 400  mg of slow - release alpha - lipoic acid or placebo , both in a lyophilized formulation , was furnished by the ra of the pediatric department .
patients were advised to melt the lyophilized solution in a glass of water and to get it twice a day at least 1 hour before lunch and dinner , for 6 months .
the compliance with the antioxidant diet was monitored by monthly dietary recalls and a full nutritional assessment was repeated , after 3 and 6 months .
both patients treated with antioxidant diet plus alpha - lipoic acid ( n = 25 , group 1 ) and patients treated with antioxidant diet plus placebo ( n = 27 , group 2 ) received the same diet plan , of 18002200  kcal per day , according to the age - group . the daily amount of carbohydrates was 60% of the total intake , with 15% of simple carbohydrates .
the prescribed amount of fiber was 40  gr per day ; fats were 26% , with a monosaturated percentage of 12 , polysaturated percentage of 4 , and saturated percentage of 8 .
the ratio of the fatty acid was 8  gr for omega-6 and 2  gr for omega-3 .
the daily amount of proteins was 14% of the total intake , with 54% of animal proteins and 46% of vegetable proteins .
no particular nutrient source to elevate the dietary orac was administered , but a list of foods with high orac content was given to each subject with detailed instruction to reach the desired assumption of orac .
a similar dietary plan was assigned to patients enrolled in the control group ( n = 19 , group 3 ) , but no orac supplementation was suggested .
prior to data analysis , all metric variables were checked for normality by using shapiro - wilk test .
we used unpaired t - test and manova ( multiple analysis of variance ) when appropriate .
results are presented as mean  standard deviation ( sd ) , or percentage , unless stated otherwise .
all the 71 patients enrolled completed the 6-month study period and no one dropped out of study before completing it .
moreover no severe side effects have been reported by either alpha - lipoic acid group or placebo .
three patients of the alpha - lipoic acid group and 2 patients of the control group reported mild abdominal pain over the first 4 weeks of the study , with no need to withdraw from the study .
baseline and 6-month clinical characteristics are summarized in table 1 for each study group . at baseline ,
the mean age of the whole group was 16.3  3.4 years , the mean of diabetes duration was 8.1  5.2 years , the mean bmi was 21  3 , and insulin requirement was 0.83  0.29  u / kg / day .
all patients enrolled in the study reported in - range levels of triglycerides , total cholesterol , ldl - cholesterol , and hdl - cholesterol per age and sex over the study period .
moreover all patients showed normal blood pressure measurements per age , sex , and height .
there was no statistical difference in all the demographic and clinical variables considered among the three study groups at baseline ( table 1 ) .
after 3 months , a significant reduction in daily insulin requirements was observed only in group 1 , both in the amount of the total daily dose ( 0.74  u / kg / day at 3 months versus 0.83  u / kg / day at baseline , p = 0.048 ) and in the percentage of bolus ( 22.0% at 3 months versus 26.3% at baseline , p = 0.047 ) ( table 1 ) .
however these data were not confirmed at 6 months , and no differences were reported among the 3 groups after 6 months of diet in terms of daily insulin requirement .
these results are supported by data collected during the nutritional assessment . indeed , dietary recall to evaluate patients ' compliance to the dietary plan showed a significant increase of orac intake at month 3 in both arms ( treatment and placebo ) but not in controls .
after 6 months , orac consumption decreased in the treatment group but not in the placebo group , as reported in table 1 .
endothelial dysfunction was tested as rhi score in all groups at baseline and after 6 months . as described in a previous study , at baseline a low rhi score
was reported in all patients enrolled , which resulted in a pathologic range ( < 1.67 ) . at 6 months , rhi score
significantly improved only in the group of patients treated with antioxidant diet plus alpha - lipoic acid ( 1.72 versus 1.4 , p = 0.045 ) , reaching a normal rhi value   ( table 1 and figure 1 ) .
the group of patients treated with antioxidant diet plus placebo reported a better rhi score at 6 months versus baseline as well , but it did not reach the significance .
no differences in rhi score were showed in the control group at 6 months versus baseline .
the analyses performed on the other clinical parameters collected showed no differences at baseline and after 3 and 6 months among the three groups : bmi , 24  h blood pressure , lipid profile , hba1c , dietary habits , and body composition resulted any different among the three groups and over the study period .
to the best of our knowledge , this is the first study analyzing the effects of a dietary supplementation in improving the endothelial dysfunction in youth with type 1 diabetes . in this randomized , placebo controlled double - blind trial
, we found a relationship between the consumption of an antioxidant diet plus alpha - lipoic acid and the improvement in endothelial dysfunction in youth with type 1 diabetes .
in particular an antioxidant diet ( 10000 orac ) plus alpha - lipoic acid ( 800  mg / day ) seems to be effective in reducing insulin requirement and daily bolus rate after 3 months and in improving endothelial dysfunction after 6 months .
the efficacy of alpha - lipoic acid in improving glucose disposal has been previously described in animal models [ 1215 ] and in type 2 diabetes adult patients [ 13 , 14 , 16 ] .
it was 1970 when lipoic acid was shown to enhance glucose uptake into rat tissue [ 17 , 18 ] .
gradually the effects of different formulas of alpha - lipoic acid were tested both in vitro and in animal models [ 16 , 19 ] confirming the ability to increase glucose uptake and to enhance the glycogen synthesis [ 16 , 19 ] .
the molecule seems to work improving insulin sensitivity by modulating the signal transduction and by increasing the glucose uptake . in patients with type 2 diabetes , an acute intravenous administration of 1000  mg of lipoic acid
moreover a 4-week oral treatment with alpha - lipoic acid successfully resulted in increasing insulin sensitivity in patients with type 2 diabetes . in our small study ,
a supplementation with 800  mg / day of alpha - lipoic acid for 3 months effectively resulted in reducing insulin requirement .
the mechanisms of action are not completely clear , since , in our knowledge , studies having pediatric type 1 diabetes patients as target are lacking .
however , as reported for type 2 diabetes , we hypothesize that alpha - lipoic acid can use nicotinamide adenine dinucleotide molecules ( nadh ) for the reduction to dihydrolipoic acid , resulting in an increased ratio of nicotinamide adenine dinucleotide ( nad+ ) to nadh and thus stimulating the glycolysis pathway . in our study
the alpha - lipoic beneficial effect on insulin requirement and thus on glucose uptake is reported at 3 months but it is not confirmed at 6 months . in our opinion this can be explained by a decreased compliance to the dietary supplementation , as confirmed by dietary recall and by data collected during the nutritional assessments .
we also demonstrated for the very first time the power of alpha - lipoic acid in improving endothelial dysfunction in youths with type 1 diabetes . in our cohort of patients ,
the prevalence of endothelial dysfunction is largely represented , despite the pediatric age and the quite good metabolic control .
data from registries has recently shown that a large proportion of the type 1 diabetic population does not meet the age - associated hba1c targets across all countries , especially in the youth age .
moreover , data recently published by lind and colleagues   showed that patients with type 1 diabetes and on - target hba1c still have a risk of death from any cause and from cardiovascular diseases more than twice the risks in the general population .
these dramatic data ask for new insights into the pathogenesis of diabetes - related complications , as well as for new therapeutic approaches .
thus , during the last decades , many studies have been conducted to investigate the role of inflammation in diabetes onset and in diabetes - related complications .
the opportunity for an early detection of endothelial dysfunction can lead to new insights into the very first steps of the cardiovascular complications . indeed
, endothelial cells play a wide spectrum of different functions , as regulating coagulation , leukocyte adhesion , and trafficking , modifying the tone of the vessel , and participating in the smooth - muscle growth . as a consequence
, many different pathways can be affected by pathologic factors , leading to endothelial dysfunction .
several mechanisms have been suggested to explain endothelial dysfunction : hyperglycemia itself , increased oxidative stress and subsequent ages production and protein kinase c and polyol - pathway activation , lower vitamin c plasma concentration , and significantly higher circulating reactive protein c levels [ 27 , 28 ] . alpha - lipoic acid is reported to be effective in most of these reactions , getting the role of antioxidant , anti - inflammatory molecule . indeed , although it is well defined as a therapy for preventing diabetic polyneuropathies and it is used in the management of diabetic peripheral neuropathy in both patients with type 1 and type 2 diabetes , alpha - lipoic acid has many biochemical functions .
it acts as metal chelator , as scavenger of free radicals , and as reducer of the oxidized forms of other antioxidant agents such as vitamins c and e and it regulates several signal transductions . in particular alpha - lipoic acid was shown to increase the activity of enos , to downregulate the expression of the cell - adhesion molecules icam-1 and vcam-1 and of mmp-9 by the inhibition of age - induced nf - kb ( nuclear factor kappa - light - chain - enhancer of activated b cells ) adhesion factors [ 14 , 22 ] . supported by this evidence
, we can speculate that a 6-month supplementation with alpha - lipoic acid 800  mg / day can positively impact endothelial dysfunction , decreasing oxidative stress and inflammation in type 1 diabetes , even in pediatrics . despite this
, these data must be carefully weighed against the lack of consensus regarding the most appropriate supplementation method for alpha - lipoic acid , dosage , and treatment duration . however , in a paper recently published a 3-month treatment with 600  mg of alpha - lipoic acid was demonstrated to provide significant beneficial changes in vegf , bfgf , mco-1 , and il-10 serum levels in type 2 diabetic patients , confirming the efficacy of low - period treatment on endothelial outcomes . moreover
, several studies demonstrated the efficacy of 600 or more mg of alpha - lipoic acid administered for few weeks or months [ 30 , 31 ] in the treatment of diabetic polyneuropathy .
the strength of our study is the evidence for the first time of a positive association between alpha - lipoic administration and decreased endothelial dysfunction in pediatric patients with type 1 diabetes .
nevertheless the trial has some limitations , and we strongly advise a larger sample and a long - term follow - up to confirm these results .
moreover , the effects of alpha - lipoic acid on glycemic control need to be investigated as well , both as glycemic variability and hypo- and hyperglycemic events .
 after evaluating the prevalence of early endothelial dysfunction , as measured by means of reactive hyperemia in adolescents with type 1 diabetes , we started a 6-month , double - blind , randomized trial to test the efficacy of an antioxidant diet (  alpha - lipoic acid supplementation ) to improve endothelial dysfunction . 
 seventy - one children and adolescents , ages 17  3.9  yrs , with type 1 diabetes since 9.5  5.3  yrs , using intensified insulin therapy , were randomized into 3 arms : ( a ) antioxidant diet 10.000 orac + alpha - lipoic acid ; ( b ) antioxidant diet 10.000 orac + placebo ; ( c ) controls . 
 bmi , blood pressure , fasting lipid profile , hba1c , insulin requirement , dietary habits , and body composition were determined in each patient . 
 an antioxidant diet significantly improved endothelial dysfunction when supplemented with alpha - lipoic acid , unlike diet with placebo or controls . 
 a significant reduction in bolus insulin was also observed . 
 we speculate that alpha - lipoic acid might have an antioxidant effect in pediatric diabetes patients by reducing insulin . 