Published online July 3, 2006
PEDIATRICS Vol. 118 No. 1 July 2006, pp. 242-253 (doi:10.1542/peds.2005-2143)
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Folate and Vitamin B6 Rapidly Normalize Endothelial Dysfunction In Children With Type 1 Diabetes Mellitus

Karen E. MacKenzie, MBChB, FRACPa,b, Esko J. Wiltshire, MD, FRACPc, Roger Gent, DMUd, Craig Hirte, BSc(Hons)e, Lino Piotto, DMUd and Jennifer J. Couper, MD, FRACPa,b

a Department of Diabetes and Endocrinology
d Department of Paediatric Ultrasonography
e Public Health Research Unit, Children, Youth, and Women's Health Service, Adelaide, Australia
b Department of Paediatrics, University of Adelaide, Adelaide, Australia
c Department of Paediatrics, Wellington School of Medicine and Health Sciences, Wellington, New Zealand


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND. Endothelial dysfunction, a precursor of vascular disease, begins early in type 1 diabetes mellitus and is associated with folate status.

METHODS. A randomized, double-blind, placebo-controlled study of folate (5 mg daily) and vitamin B6 (100 mg daily) in 124 children with type 1 diabetes determined the immediate and 8-week effects of these vitamins, alone and in combination, on endothelial function. Endothelial function, assessed as flow-mediated dilation and glyceryltrinitrate-induced dilation with high-resolution ultrasound of the brachial artery, was measured at baseline, at 2 and 4 hours after the first dose (n = 35), and at 4 and 8 weeks of treatment (n = 122).

RESULTS. Flow-mediated dilation normalized in all treatment groups. From baseline to 8 weeks, flow-mediated dilation improved with folate from 2.6% ± 4.3% (mean ± SD) to 9.7% ± 6.0%, with vitamin B6 from 3.5% ± 4.0% to 8.3% ± 4.2%, and with folate/vitamin B6 from 2.8% ± 3.5% to 10.5% ± 4.4%. This improvement in flow-mediated dilation occurred within 2 hours and was maintained at 8 weeks for each treatment. Flow-mediated dilation in the placebo group, and glyceryltrinitrate-induced dilation in all groups, did not change. Increases in serum folate, red cell folate, and serum vitamin B6 levels related to increases in flow-mediated dilation. Improvement in flow-mediated dilation was independent of changes in total plasma homocyst(e)ine, glucose, hemoglobin A1c, and high-sensitivity C-reactive protein levels. Baseline red cell folate levels and baseline diastolic blood pressure were related inversely to improvement in flow-mediated dilation. Serum triglyceride and low-density lipoprotein cholesterol inversely related to baseline flow-mediated dilation.

CONCLUSIONS. High-dose folate and vitamin B6 normalized endothelial dysfunction in children with type 1 diabetes. This effect was maintained over 8 weeks, with no additional benefit from combination treatment.

Key Words: type 1 diabetes • endothelial function • folate • pyridoxine • randomized • controlled trial

Abbreviations: FMD—flow-mediated dilation • HbA1c—hemoglobin A1c • Hs-CRP—high-sensitivity C-reactive protein • LDL—low-density lipoprotein • HDL—high-density lipoprotein • RCF—red blood cell folate • WCH—Women's and Children's Hospital

Prevention of the vascular complications of diabetes mellitus requires strategies that begin early in the course of the disease. Endothelial dysfunction, a fundamental event in the development of atherosclerosis,1 occurs early in type 1 diabetes, before clinically detectable atherosclerotic disease.2 It is critical to the pathogenesis of microvascular and macrovascular complications of diabetes.3

Endothelial dysfunction of the coronary arteries predicts atherosclerotic disease progression and cardiovascular events.4 Endothelial function of the brachial artery can be assessed noninvasively with flow-mediated dilation (FMD), which measures changes in vessel diameter in response to increased arterial flow (endothelium-dependent vasodilation), and glyceryltrinitrate-induced vasodilation, which measures changes in vessel diameter in response to glyceryltrinitrate, which acts directly on smooth muscle (endothelium-independent vasodilation).5 Brachial artery FMD correlates well with coronary endothelial function6–8 and carotid intimal medial thickness.9 It is a sensitive and specific screening test to predict coronary artery disease10 and provides a surrogate measure of coronary endothelial function. FMD can therefore assess the reversibility of endothelial dysfunction among asymptomatic subjects at high risk of arterial disease and, being safe and noninvasive, is suitable for use among children. The ability to detect and to monitor changes in FMD among children who are at high risk of vascular disease in adulthood but for whom clinically detectable disease is not evident may identify interventions that can improve long-term vascular health.

Hyperglycemia,11–13 insulin,14 low-density lipoprotein (LDL) cholesterol,2 and homocyst(e)ine15 all contribute to endothelial dysfunction in type 1 diabetes. Folate improved endothelial function among adults with the risk factors of type 2 diabetes,16 high LDL cholesterol levels,17, 18 hyperhomocyst(e)inemia,19–21 and cardiovascular disease.22–24 In children, folate improves endothelial function associated with chronic renal failure.25

Treatment with the combination of folate and vitamin B6 decreases markers of endothelial activation.26, 27 However, there is limited literature examining the effect of B6 alone on the endothelium. Vitamin B6 improved endothelial function among cardiac transplant recipients.28 There are no data examining the effect of supplemental vitamin B6 in type 1 diabetes or among children at risk of vascular disease.

We showed that endothelial dysfunction is common in children with type 1 diabetes and relates to folate status29 despite higher serum and red cell folate (RCF) levels and lower total plasma homocyst(e)ine levels than those of healthy control subjects.30, 31 Even with these higher folate levels, we showed, in a small, pilot, crossover study, that folate supplementation improves endothelial function in children with type 1 diabetes.32

With a larger group of children with type 1 diabetes, we aimed to examine the immediate effects of folate and vitamin B6 on endothelial function and the effects of folate and vitamin B6, alone or in combination, on endothelial function over 8 weeks. We also aimed to study the determinants of endothelial function among these children.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Participants
Subjects with type 1 diabetes were recruited consecutively from the diabetes clinic at the Women's and Children's Hospital (WCH) (Adelaide, Australia) between November 2002 and November 2003. A power calculation based on results from our previous study32 indicated that 30 subjects in each treatment group would have 74% power to detect a difference in FMD results with treatment of 2.0% ± 3%. To allow for potential difficulties in interpretation of FMD, 124 subjects were recruited.

Subjects were excluded before recruitment if they were known to have celiac disease (which may reduce absorption of folate and/or vitamin B6), were active smokers, or were taking supplemental vitamins, including multivitamins. All subjects were screened for celiac antibodies (antigliadin, endomysial, total IgA), thyroid function, and vitamin B12 deficiency, which precludes folate treatment. Subjects had had diabetes for ≥1 year, were normotensive, and had no clinically detectable microvascular disease, as assessed with overnight urinary albumin excretion and direct fundoscopy performed through dilated pupils by experienced pediatric ophthalmologists. At the time of testing, subjects were well, without ketosis or hypoglycemia in the previous 24 hours.

All parents and subjects received written information and gave informed written consent. Contact was maintained with the subjects to ensure early reporting of adverse events that would have resulted in the subject being withdrawn from the study. No adverse events were reported. To aid adherence to the study and daily tablet taking, telephone calls to subjects were made at weeks 3 and 7.

Study Design
The study had a randomized, double-blind, placebo-controlled design. The 4 study groups received (1) 5 mg of folate (Sigma Pharmaceuticals, Melbourne, Australia) and matched placebo (Sigma Pharmaceuticals), (2) 100 mg of vitamin B6 (Rhone-Poulenc-Rorer Pharmaceuticals, Sydney, Australia) and matched placebo, (3) 5 mg of folate and 100 mg of vitamin B6, and (4) 2 placebo tablets. After recruitment by 1 author (K.E.M.), subjects were assigned a unique identifying number. Randomization, with this identifying number, was then performed by using a Fischer table by the pharmacy department at WCH. According to randomization, subjects were allocated to 1 of the 4 study groups described above, to receive 2 tablets each day for 8 weeks. The tablets were provided, in 2 identical bottles labeled A and B, by the pharmacy department at WCH. The 2 bottles were supplied in a paper bag labeled only with the subjects' unique identifying number. Subjects were instructed to take 1 tablet from each bottle daily and to take both tablets at the same time each day, preferably in the morning. Subjects were also instructed not to take any additional vitamin supplements during the study period.

Subjects underwent 3 morning assessments (7:30 to 8:30 AM), at baseline, 4 weeks, and 8 weeks. At each assessment, subjects fasted and withheld morning insulin. Subjects were also advised not to take the study tablets before the morning assessments. Clinical data were collected, brachial artery responses to FMD and glyceryltrinitrate were assessed, and venous blood samples were collected at each assessment. Blood was collected at baseline for vitamin B12, serum cotinine, and lipid measurements. Blood was collected at each assessment for glucose, hemoglobin A1c (HbA1c), high-sensitivity C-reactive protein (Hs-CRP), RCF, serum folate, vitamin B6, and total plasma homocyst(e)ine measurements.

Thirty-five subjects also were studied for 4 hours on their first visit, to determine the immediate effects of folate and vitamin B6 (Fig 1). Baseline studies were performed, subjects were instructed to take 1 of the 2 tablets (either A or B) according to the randomization, and assessments were repeated at 2 and 4 hours. Subjects were recumbent during the 4 hours and remained in a temperature-controlled environment (22–24°C). A carbohydrate-based, low-fat, low-protein, caffeine-free breakfast was provided, and insulin was administered subcutaneously immediately after the baseline assessment. The meal was completed within 30 minutes after the baseline assessment.


Figure 1
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  		FIGURE 1 Trial profile.

 
Outcome Measurements
Noninvasive Measurement of Endothelial Function
The primary outcome measure was assessment of endothelial function by FMD (endothelium-dependent) and glyceryltrinitrate-induced vasodilation (endothelium-independent), as reported previously.5, 29 With this method, FMD and the FMD/glyceryltrinitrate-induced dilation ratio provide the best measure of endothelial function,5, 29 with glyceryltrinitrate-induced dilation acting as a positive control measurement of vascular reactivity for each study. FMD is dependent on endogenous nitric oxide release.33

Ultrasound studies were performed by 2 experienced senior pediatric vascular ultrasonographers. The diameter of the right brachial artery, 2 to 15 cm above the elbow and proximal to the placement of the blood pressure cuff, was measured in longitudinal section from 2-dimensional ultrasound images by using a standard HDI 3000 ultrasound system (Advanced Technology Laboratories, Bothel, WA) and a 10.0-MHz, linear-array transducer. A suitable site for vessel imaging with reproducible markers was selected, to ensure that measurement was at the same place for each scan. The transmit (focus) zone was set to the depth of the vessel. Depth and gain settings were set to optimize images of the lumen/arterial wall interface. Instrument parameters were not changed in any study. An electrocardiogram was recorded with the ultrasound images. The first scan was taken at rest. Reactive hyperemia was induced by occluding arterial blood flow by using a sphygmomanometer with an adult-size cuff, inflated to 250 mm Hg, for 4 minutes. Arterial flow velocity was measured with a pulsed Doppler signal at 60° to the vessel. A scan of artery diameter was taken for 30 seconds before and 120 seconds after cuff deflation. FMD is the greatest increase in end diastolic diameter after cuff release. In children, this occurs 45 to 120 seconds after cuff deflation. Fifteen minutes was allowed for vessel recovery, and then another resting scan was performed. Glyceryltrinitrate (400 µg of Nitrolingual spray; Rhone-Poulenc-Rorer Pharmaceuticals) was administered sublingually, and a scan was recorded 4 minutes later.

Images were recorded on high-quality VHS videotape. At completion of the study, scans were analyzed by 2 investigators (K.E.M., E.J.W.), who were blinded to both the stage of the study and the intervention group. Vessel measurements, measured with electronic calipers, were incident with the electrocardiogram R wave (ie, at end diastole), taken over 4 cardiac cycles, and averaged. Measurements were expressed as percentages of the first control scan, after reactive hyperemia and after glyceryltrinitrate. Brachial artery FMD was calculated as the maximal change in diameter from baseline, as a percentage. Our coefficient of variation for 20 control subjects was 3.9% for FMD and 4.0% for glyceryltrinitrate-induced dilation.29

Biochemical Assays
Glucose was measured with hexokinase spectrophotometry (Synchron CX5CE system; Beckman, Fullerton, CA). HbA1c was measured with latex immunoagglutination inhibition (DCA 2000, HbA1c reagent kit; Bayer, Toronto, Canada). The nondiabetic normal range is 4.0% to 6.0%. This method has been correlated with high performance liquid chromatography in our laboratory (r = 0.97). The high performance liquid chromatography method is standardized with Diabetes Control and Complications Trial control sera. Total plasma homocyst(e)ine, serum folate, RCF, and vitamin B12 levels were measured with the Abbott IMX analyzer (Abbott AxSYM), as reported previously.31 The serum folate and RCF level normal ranges are 3.4 to 15.0 µg/L (7.7–33.9 nmol/L) and 168 to 388 µg/L (380–878 nmol/L), respectively. Interassay coefficients of variation were 5.5%. Vitamin B6 status was determined as percentage activation of red blood cell aspartate aminotransferase, a pyridoxal phosphate enzyme activation test; normal values are <63%. Hs-CRP was measured with rate nephelometry and rate turbidimetry, with the Beckman Coulter IMMAGE immunochemistry system (Beckman Coulter). Lipids, including total, high-density lipoprotein (HDL), and LDL cholesterol and triglycerides, were measured spectrophotometrically, with a commercial, timed, end-point method with the Synchron CX5CE system (Beckman). Serum cotinine levels were measured with a cotinine microplate enzyme immunoassay (STC Technologies, Victoria, Australia).

Ethics
The study was approved by the WCH Human Research Ethics Committee.

Statistical Analyses
One-way analysis of variance was used to compare continuous baseline variables between treatment groups, with Q-Q plots confirming the assumption of normality for both FMD and glyceryltrinitrate-induced dilation. Kruskal-Wallis tests were also used and gave similar results, confirming the effect shown through analysis of variance. {chi}2 tests were used to compare categorical baseline variables between treatment groups. Spearman correlations were calculated to assess the association between baseline FMD and the other baseline variables and to assess the association between baseline glyceryltrinitrate-induced dilation and the other baseline variables. Multivariate analysis of variance models for FMD and glyceryltrinitrate-induced dilation, followed by a backward selection method, were used to determine the variables with stronger independent association with FMD or glyceryltrinitrate-induced dilation. Linear mixed models with simple contrasts were used to assess separately the change in FMD over 4 hours and 8 weeks. Spearman correlations were calculated to assess the association between the change in FMD over the 8-week period and both the baseline measurement and the change over the 8-week period for other variables. Similarly, Spearman correlations were calculated for the change over the 4-hour period. Full disclosure of all tests performed and precise P values are reported.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Study Group
A total of 124 of 162 subjects who were approached consecutively agreed to enter the study. Subjects declined to enter the study predominantly on the basis of distance from the hospital and occasionally on the basis of multivitamin usage or smoking history; some gave no reason. Of the 124 who entered the study, 35 subjects completed the 4-hour immediate-effects study, all but 1 of whom continued into the 8-week study; there was 1 withdrawal after the 4-week assessment. A total of 122 subjects completed the study at 8 weeks (Fig 1).

Baseline Characteristics
Baseline characteristics are shown in Tables 1 and 2. There were no significant differences in the clinical characteristics between the groups (Table 1). The placebo group did, however, have a significantly higher FMD at baseline, compared with the 3 intervention groups (Table 2) (see "Discussion"). All subjects had normal folate status determined with serum folate and RCF levels. Four were deficient in vitamin B6 as determined by percent pyridoxal phosphate activation (median: 71.4%; range: 63.8%–93.2%; normal range: <63%).


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  		TABLE 1 Baseline Clinical Characteristics

 

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  		TABLE 2 Baseline Laboratory Results

 
Five subjects with positive celiac antibodies at baseline had celiac disease confirmed through small-bowel biopsy after completion of the study. One subject was diagnosed as having Graves' disease. These subjects were included in the analysis, because exclusion did not alter the final results.

None gave a history of smoking; however, 5 subjects had elevated serum cotinine levels (>28 nmol/L). Two, with levels consistent with passive smoking (<115 nmol//L), were randomly assigned to the placebo group. Three, with levels within the active smoking range (115–5700 nmol/L), were randomly assigned to the folate/vitamin B6 group (2 subjects) or the folate group (1 subject). Again, these subjects were included in the analysis because exclusion did not alter the final results.

For the 8-week study, tablet counts were 91% complete. There was no difference in tablet counts between the groups (P = .6).

FMD
All scans were of sufficient technical quality to be analyzed. The coefficient of variation in the placebo group throughout the study was 2.5%, indicating highly reproducible results.

Baseline (week 0) FMD was associated inversely with levels of triglycerides (r = –0.25; P = .007) and LDL cholesterol (r = –0.18; P = .05). The multivariate analysis of variance models showed that resting vessel diameter (ß = –.39; P < .001) and LDL cholesterol levels (ß = –.21; P = .02) influenced baseline FMD independently. Age, duration of diabetes, blood pressure, insulin dose, serum folate level, RCF level, vitamin B6 status, total plasma homocyst(e)ine level, glucose level, HbA1c level, Hs-CRP level, and total and HDL cholesterol levels were not correlated significantly with baseline FMD.

There was no significant difference in mean resting vessel diameter within each group over the duration of the study, in either the immediate-effects or 8-week study. In the immediate-effects study, there was no significant difference in mean resting vessel diameter between groups at each time point. There was no significant difference in mean resting vessel diameter in the total group over the duration of the study in either the immediate-effects study (P = .8) or the 8-week study (P = .8) (Tables 3 and 4). There was no significant difference in stimulus for FMD, ie, percentage increase in arterial flow velocity, within each group over the duration of each study or between groups at each time point, and there was no difference in stimulus for FMD in any group over the duration of the study in either the immediate-effects study (P = .5) or the 8-week study (P = .4) (Tables 5 and 6).


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  		TABLE 3 Resting Vessel Diameters in the Immediate-Effects Study

 

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  		TABLE 4 Resting Vessel Diameters in the 8-Week Study

 

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  		TABLE 5 Stimulus for FMD (Increase in Flow) for the Immediate-Effects Study

 

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  		TABLE 6 Stimulus for FMD (Increase in Flow) for the 8-Week Study

 
Immediate Effects of Folate and Vitamin B6
At 2 hours, folate increased FMD significantly, from 1.88% ± 3.93% (mean ± SD) to 10.46% ± 5.12% (P < .001) (Fig 2). There was no additional increase in FMD at 4 hours (P = .78). At 2 hours, vitamin B6 increased FMD significantly, from 4.70% ± 4.04% (mean ± SD) to 8.36% ± 4.31% (P = .007) (Fig 2). There was no additional increase in FMD at 4 hours (P = .59). There was no significant change in FMD in the placebo group over 4 hours (from 6.16% ± 3.56% to 5.90% ± 4.34%; P = .41) (Fig 2). There was no significant change in glyceryltrinitrate-induced dilation at 2 or 4 hours in any group.


Figure 2
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  		FIGURE 2 Immediate effects of folate and vitamin B6 on FMD (mean ± SE). Green squares indicate placebo group, with no change in FMD over 4 hours (P = .41); red diamonds, folate group, with a significant change in FMD at 2 hours (P < .001) that was maintained at 4 hours; blue triangles, vitamin B6 group, with a significant change in FMD at 2 hours (P = .007) that was maintained at 4 hours.

 
For all subjects in the folate group, serum folate levels increased significantly to above the normal range (3.4–15.0 µg/L), from 10.7 ± 4.6 to 19.2 ± 0.57 µg/L, within 4 hours (P < .001). Serum folate levels increased rapidly to supraphysiologic values (57.6–465.0 µg/L) after the oral folate dose for 7 subjects in the folate group. RCF levels also increased significantly, from 451.3 ± 154.9 to 623.4 ± 179.1 µg/L, within 4 hours (P < .001) in the folate group. There was no significant change in serum folate and RCF levels from baseline values in either the placebo group (P = .06 and P = .9, respectively) or the vitamin B6 group (P = .1 and P = .6, respectively). There was a significant improvement in vitamin B6 status in the vitamin B6 group, from 48.0% ± 4.85% to 43.6% ± 4.53% pyridoxal phosphate activation, within 4 hours (P = .004). There was no significant change in vitamin B6 levels from baseline values in either the placebo group (P = .3) or the folate group (P = .7).

Eight-Week Effects of Folate and Vitamin B6 on Endothelial Function
FMD at the end of 8 weeks was significantly greater in all 3 intervention groups than in the placebo group (P < .001). The increase in FMD was seen at 4 weeks and was maintained at 8 weeks in all intervention groups. After 4 weeks, folate (5 mg daily) increased FMD from a baseline of 2.6% ± 4.27% (mean ± SD) to 9.67% ± 6.00% (P < .001); at 8 weeks, there was no additional increase in FMD (P = .62) (Fig 3). After 4 weeks, vitamin B6 increased FMD from a baseline of 3.50% ± 4.02% (mean ± SD) to 8.31% ± 4.21% (P < .001); at 8 weeks, there was no additional increase in FMD (P = .11) (Fig 3). After 4 weeks, the combination of folate and vitamin B6 increased FMD from a baseline of 2.79% ± 3.45% (mean ± SD) to 10.48% ± 4.39% (P < .001); at 8 weeks, there was no additional increase in FMD (P = .77) (Fig 3). There was no significant change in FMD in the placebo group over the 8 weeks (mean ± SD: 6.16% ± 4.10% to 5.44% ± 3.37%; P = .09) (Fig 3). There was no significant change in glyceryltrinitrate-induced dilation at 4 or 8 weeks in any group.


Figure 3
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  		FIGURE 3 Effects of folate, vitamin B6, and folate/vitamin B6 combination on FMD (mean ± SE) over 8 weeks. Green squares indicate placebo group, with no change in FMD over 8 weeks (P = .09); red diamonds, folate group, with a significant change in FMD at 4 weeks (P < .001) that was maintained at 8 weeks; blue triangles, vitamin B6 group, with a significant change in FMD at 4 weeks (P < .001) that was maintained at 8 weeks; yellow crosses, folate/vitamin B6 group, with a significant change in FMD (P < .001) at 4 weeks that was maintained at 8 weeks.

 
There was no significant difference in the increase in FMD between subjects who received folate (5 mg) daily for 8 weeks, vitamin B6 (100 mg) daily for 8 weeks, or both folate (5 mg) and vitamin B6 (100 mg) daily for 8 weeks (P = .51) (Fig 3). There was no significant difference in FMD at any time point from 2 hours to 8 weeks in any of the 3 intervention groups.

For all subjects in the folate and folate/vitamin B6 groups, serum folate levels increased significantly to above the normal range (3.4–15.0 µg/L), from 12.3 ± 4.4 to 15.9 ± 3.1 µg/L and from 12.5 ± 2.7 to 16.5 ± 2.3 µg/L, respectively, over the 8 weeks (P < .001). RCF levels also increased significantly in the folate and folate/vitamin B6 groups, from 456.0 ± 129.1 to 704.4 ± 163.3 µg/L and from 470.1 ± 130.6 to 751.7 ± 107.0 µg/L respectively, over the 8 weeks (P < .001). There were significant decreases in both serum folate and RCF levels in the vitamin B6 group over the 8 weeks, from 12.8 ± 3.1 to 10.8 ± 3.6 µg/L (P < .001) and from 473.5 ± 158.9 to 426.4 ± 155.9 µg/L (P = .01), respectively; despite this, both serum folate and RCF levels remained within the normal range in this group. There was no significant change from baseline values in serum folate and RCF levels in the placebo group (P = .2 and P = .9, respectively). There was a significant improvement in vitamin B6 status in both the vitamin B6 and folate/vitamin B6 groups, from 48.3% ± 6.8% to 19.4% ± 15.8% pyridoxal phosphate activation and from 48.3% ± 8.0% to 20.3% ± 10.1% pyridoxal phosphate activation, respectively, over the 8 weeks (P < .001). There was no significant change in vitamin B6 levels from baseline values in either the placebo group (P = .4) or the folate group (P = .3).

There was no significant difference in FMD between the 3 intervention groups (the subjects receiving folate, vitamin B6, or folate/vitamin B6) and a previously studied group of 20 healthy control children without diabetes29 at 8 weeks (P = .79) (Fig 4). The age-matched, healthy, control children were studied under identical conditions, with the same ultrasonographer and the same equipment, and results were assessed by the same blinded observer.29 There was no change from baseline values for the subjects receiving placebo at 8 weeks (P = .25).


Figure 4
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  		FIGURE 4 Box plots for FMD at 8 weeks. The boxes represent the interquartile range, the dark horizontal lines represent the median, the whiskers represent the maximal and minimal values, and outliers are shown. There was no significant difference in FMD between the 4 groups of previously studied healthy control subjects (n = 20)29 (white) and subjects receiving folate (n = 31) (red), vitamin B6 (n = 31) (blue), and folate/vitamin B6 (n = 30) (yellow) (P = .791, 1-way analysis of variance), at 8 weeks. The placebo group (n = 30) (green) had significantly lower FMD than all groups at 8 weeks (P < .001). There was no change from baseline FMD to FMD at 8 weeks for the subjects receiving placebo (P = .254). T1DM indicates type 1 diabetes mellitus.

 
The change in FMD over 8 weeks correlated with increases in RCF levels (r = 0.45; P < .001) and serum folate levels (r = 0.27; P = .004) and improvement in vitamin B6 status (r = –0.24; P = .01). At 4 hours, the change in FMD also correlated with an increase in RCF levels (r = 0.42; P = .01) and improvement in vitamin B6 status (r = 0.35; P = .05) but not with a change in serum folate levels (r = 0.26; P = .18). Changes in FMD over 4 hours and 8 weeks did not correlate with total plasma homocyst(e)ine, glucose, HbA1c, or Hs-CRP levels.

For the 123 subjects, the baseline RCF level (r = –0.18; P = .04) and baseline diastolic blood pressure (r = –0.22; P = .02) correlated inversely and weakly with increases in FMD over 8 weeks. Changes in FMD at 2 and 4 hours also correlated with baseline vitamin B6 status (r = –0.38; P = .03) and insulin dose (r = 0.36; P = .03). Age, duration of diabetes, and baseline serum folate, total plasma homocyst(e)ine, glucose, HbA1c, Hs-CRP, total, HDL, and LDL cholesterol, and triglyceride levels did not correlate with changes in FMD at either 4 hours or 8 weeks.

The baseline glyceryltrinitrate-induced dilation in the placebo group was significantly higher than that the treatment groups (P = .05). Baseline glyceryltrinitrate-induced dilation correlated with baseline FMD (r = 0.35; P < .001), resting vessel diameter (r = –0.59; P < .001), age (r = –0.35; P < .001), systolic blood pressure (r = –0.47; P < .001), and insulin dose (r = –0.32; P < .001). Glyceryltrinitrate-induced dilation did not change significantly over 4 hours or over 8 weeks in any group (Fig 5). Baseline glyceryltrinitrate-induced dilation also correlated weakly with BMI (r = –0.18; P = .05) and HDL cholesterol levels (r = 0.2; P = .03). Multivariate analysis of variance models showed that resting vessel diameter (ß = –.52; P < .001), systolic blood pressure (ß = –.20; P = .01), and insulin dose (ß = –.15; P = .04) influenced baseline glyceryltrinitrate-induced dilation independently.


Figure 5
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  		FIGURE 5 Effects of folate, vitamin B6, and folate/vitamin B6 combination on glyceryltrinitrate (GTN)-induced dilation (mean ± SE) over 8 weeks. Green squares indicate placebo group, with no change in glyceryltrinitrate-induced dilation over 8 weeks (P = .1); red diamonds, folate group, with no change in glyceryltrinitrate-induced dilation over 8 weeks (P = .17); blue triangles, vitamin B6 group, with no change in glyceryltrinitrate-induced dilation over 8 weeks (P = .6); yellow crosses, folate/vitamin B6 group, with no significant change in glyceryltrinitrate-induced dilation (P = .2) over 8 weeks.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Children with type 1 diabetes mellitus have severe endothelial dysfunction. We have shown for the first time that, among children with type 1 diabetes and normal folate status, both high-dose folate and high-dose vitamin B6 normalize endothelial function within 2 hours, an effect that is maintained over 8 weeks with continued treatment with high-dose folate and/or vitamin B6. The combination of folate and vitamin B6 did not further improve endothelial function, compared with folate or vitamin B6 alone. Glyceryltrinitrate-induced vasodilation (endothelium-independent vasodilation) was not altered by folate and/or vitamin B6, which indicated no effect on smooth muscle function.

The placebo group had significantly higher baseline FMD (P < .001) and glyceryltrinitrate-induced dilation (P = .05) values than did the 3 intervention groups. Despite this, all 3 treatment groups had significantly higher FMD values than did the placebo group (P < .001) after intervention, which indicated the effectiveness of folate and vitamin B6 in improving endothelial function. Subjects were assigned randomly at entry into the study. We did not perform randomization at the beginning of the study on the basis of FMD, glyceryltrinitrate-induced dilation, or the characteristics of the group. Therefore, the variation seen between the groups in baseline values is likely to be a result of the randomization process. The reproducibility of the FMD in the placebo group (coefficient of variation: 2.5%) indicates that there is no discrepancy with measurement variation and that the differences in baseline FMD and glyceryltrinitrate-induced dilation values are the result of randomization. The FMD and glyceryltrinitrate-induced dilation remained constant throughout the study for the placebo group.

Screening with serum cotinine measurements identified 3 subjects as active cigarette smokers, all of whom had significant and severe endothelial dysfunction at baseline. Exclusion of these subjects, who were assigned randomly to the folate (1 subject) and folate/vitamin B6 (2 subjects) treatment groups, did not alter the final results, because all responded well to treatment. Folate is recognized to improve endothelial function among otherwise healthy cigarette smokers.34

Correlates of baseline FMD and predictors of FMD response to folate and vitamin B6 included known cardiovascular risk factors (LDL cholesterol levels, triglyceride levels, and diastolic blood pressure), as well as folate and vitamin B6 status at baseline and throughout the study. Although all subjects had normal folate status and all except 4 had normal vitamin B6 status, lower vitamin levels at baseline and greater improvement in vitamin levels with treatment predicted improvement in endothelial function.

The mechanism underlying the improvement in endothelial function with folate has been attributed, until recently, to reductions in homocyst(e)ine levels that occur with folate or other vitamin B therapy. We demonstrated a direct effect of folate in improving endothelial function independent of decreasing total plasma homocyst(e)ine levels, which supports several studies that also demonstrated benefits of folate independent of total plasma homocyst(e)ine levels.16–18, 23, 32 Folate may improve endothelial function through several mechanisms, including antioxidant effects, effects on cofactor availability through increases in the endogenous regeneration of tetrahydrobiopterin (an essential cofactor for endothelial nitric oxide synthase), and/or direct interactions with endothelial nitric oxide synthase to reduce superoxide production and to enhance nitric oxide production.17, 35 Endothelial function measured as FMD is a nitric oxide-mediated process.33, 36 Hyperglycemia affects nitric oxide availability by causing nitric oxide degradation and uncoupling of endothelial nitric oxide synthase.37 Folate restores the function of "uncoupled" endothelial nitric oxide synthase and enhances the endothelial production of nitric oxide.35

Folate fortification of grains, which provides an additional 70 to 120 µg of folate per day, decreases total plasma homocyst(e)ine levels and increases folate levels.38 Although this may correspond to a lower cardiovascular risk,39 low folate doses (ie, 0.4 mg/day) have not improved endothelial function among healthy subjects.40 The rapid efficacy of high-dose folate (5 mg) in this study prompts a dose-response determination of the effect of folate on endothelial function.

High-dose folate improved endothelial function among children with type 1 diabetes in our recent small crossover trial.32 In a comparison of these studies, FMD improved but was not normalized in the crossover trial, the crossover study group was smaller, and subjects had poorer metabolic control, longer duration of diabetes, and lower RCF levels both at baseline and at the study end points. Compliance in this current study was ensured more rigorously with regular telephone contact and a shorter interval between visits.

This is the first study of vitamin B6 supplementation among children with type 1 diabetes. It is also the first to examine the immediate effect of vitamin B6 on endothelial function. There is indirect evidence that supplemental vitamin B6 may be beneficial in type 1 diabetes. Vitamin B6 levels are lower among children and adults with type 1 diabetes.41, 42 In experimental diabetes, vitamin B6 utilization and requirements are higher.43 Vitamin B6 deficiency confers an independent risk of cardiovascular, cerebrovascular, and peripheral vascular disease.44–47 Restoring vitamin B6 status for children with type 1 diabetes has a beneficial effect on the endothelium; the mechanism beyond homocyst(e)ine level decrease is not yet explained and warrants additional investigation. Concerns regarding peripheral neuropathy with vitamin B6 therapy are controversial,48, 49 and no adverse effects were reported during this study or in 24 months of follow-up monitoring.

Levels of Hs-CRP, a marker of the inflammatory state associated with atherosclerosis,50, 51 did not correlate with FMD, despite this relationship being described for adults with type 1 diabetes52 and for healthy children,53 and Hs-CRP levels did not correlate with the improvement in endothelial function, consistent with other intervention studies.54 This is likely related to the variability in Hs-CRP levels and the need for subject numbers larger than those seen in intervention trials.

We have confirmed the immediate and sustained beneficial effects of folate and vitamin B6 on endothelial function among children with type 1 diabetes. The possibility of long-term benefits of folate and vitamin B6 therapy on vascular disease progression is an exciting prospect. Despite the recent disappointing lack of benefit of high-dose folate, vitamin B6, and vitamin B12 on vascular outcomes among adults with established vascular disease,55 children at high risk of vascular disease in later life but without clinically evident disease should be amenable to long-term benefits of such treatment. Effective intervention with an apparently safe, inexpensive adjunct such as folate or vitamin B6 at an early stage in childhood, in addition to optimization of metabolic control, could have a major impact on long-term diabetic vascular complications.


   ACKNOWLEDGMENTS
 
Dr MacKenzie was supported by a fellowship from the Juvenile Diabetes Research Foundation of Australia through the Royal Australasian College of Physicians. The work was supported by grants from Channel 7, Australia, and the WCH Research Foundation.


   FOOTNOTES
 
Accepted Feb 15, 2006.

Address correspondence to Karen E. MacKenzie, MBChB, FRACP, Department of Paediatrics, Christchurch Hospital, Rolleston Avenue, Christchurch, New Zealand. E-mail: karen.mackenzie{at}adelaide.edu.au

The sponsors of the study had no role in the design of the study, the collection, analysis, and interpretation of the data, the writing of the report, or the decision to submit the report for publication.

The authors have indicated they have no financial relationships relevant to this article to disclose.


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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics

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