Biochemical effects.

Neither L-arginine or atorvastatin therapy had any significant effect on fasting glucose concentration, insulin requirements or overall diabetic control as determined by fructosamine levels. In the subjects randomized to L-arginine, plasma arginine increased from 67 ± 15 to 159 ± 84 pmol/1 (P < 0.001). L-Arginine had no significant effect on plasma levels of any other amino acid, cholesterol or triglyceride levels. In the 37 subjects treated with atorvastatin, total, LDL and HDL cholesterol and triglyceride levels decreased by a mean of 33.3 ± 9.4% (P < 0.001), 48.3 ± 10.0% (P < 0.001), 6.1 ± 21.3% (P = 0.04) and 12.1 ± 25.6% (P = 0.03) respectively (Table 5.3.3).

Table 5.3.1 Baseline subject characteristics

T r e a t m e n t g r o u p s

Placebo L-Arginine Atorvastatin

L-Arginine + Atorvastatin No (%male) 22 (77) 21 (72) 21(53) 20 (55) Age 34.9 ±7.9 35.0 ±6.4 33.7 ±6.1 33.3 ±6.8 Duration of diabetes (years) 16.5 ±9.6 15.0 ±9.2 15.1 ±8.5 16.2 ± 10.2 Fructosamine (mmoI/1) 408 ± 73 374 ± 75 427 ± 64 414 ± 6 4 Glucose (mmol/l) 10.8 ±6.2 9.9 ±6.1 10.6 ±5.1 11.4±5.1

Daily insulin (lU) 52.8 ± 17.4 64.2 ± 22.5 53.3 ±25.6 50.5 ± 15.6

Arginine (pmol/l) 57.5 ±15.6 68.9 ± 17.6 70.1 ±28.6 65.2 ± 12.6

Total cholesterol (mmol/l) 4.70 ± 1.07 4.78 ±0.80 4.92 ± 1.07 5.08 ±0.86

LDL cholesterol (mmol/l) 2.82 ± 1.0 2.95 ±0.81 3.08 ±0.92 3.02 ±0.82 vonWillebrand factor (umol/1) 1.09±0.43 1.20±0.40 1.20±0.36 1.27±0.56 Fibrinogen (umol/1) 2.48±0.39 3.19±1.33 2.99±0.72 3.02±0.57 P A Il (umol/1) 7.18±6.97 5.78±5.46 5.06±3.89 6.04±3.62 HDL cholesterol (mmol/l) 1.45 ±0.33 1.34 ±0.46 1.47 ±0.37 1.62 ±0.62 Triglycerides (mmol/l) 0.93 ±0.51 1.09 ± 1.08 0.83 ± 0.44 0.97 ± 0.34 Resting vessel size (mm) 3.93 ±0.47 4.05 ± 0.65 3.69 ±0.70 3.77 ±0.73

FMD (%) 3.49 ±2.99 3.33 ±3.03 3.65 ±3.20 3.01 ±3.32

GTN-MD (%) 17.12 ±8.49 16.44 ±7.49 19.58 ±8.00 16.07 ±7.49

Resting blood flow

(mls/min) 83.0 ±48.0 110.6 ±69.7 65.9 ±53.0 86.0 ±61.1

Hyperemic blood flow

(AUC) 75.7 ±26.5 89.3 ±41.9 69.8 ± 29.0 78.6 ±35.7

PAIl = plasminogen activator inhibitor-1, FMD = flow mediated dilation, GTN-MD = glyceryl trinitrate mediated dilation, AUC = area under the time/flow curve for hyperemic blood flow over the first 20 seconds following release of the tourniquet.

Table 5.3.2. Withdrawals after randomization Study Sex

No

Age Treatm ent group Reason for w ithdraw al

1 Female 22 Atorvastatin Ketoacidosis following routine menisectomy

40 Female 33 L-arginine Lost to follow up 42 Female 36 Atorvastatin +

L-arginine

Lost to follow up

49 Male 41 Placebo Lost to follow up

53 Male 35 Atorvastatin Dizziness

55 Female 34 Atorvastatin Episodes o f hypoglycaemia 78 Female 38 Atorvastatin Lost to follow up

S.3.3.3 Vascular Function.

Baseline vessel size, resting blood flow, reactive hyperemic blood flow, FMD and dilatation to GTN were well matched between treatment groups (Table 5.3.1), For the whole cohort, FMD and dilatation to GTN were 3.4 ± 3.1% and 17.3 ± 7.9% respectively. There was a significant correlation between baseline FMD and the area under the curve of reactive hyperemic blood flow velocity over the first 20 seconds after release of the tourniquet (r = 0.23, P = 0.04) but not with peak hyperaemic blood flow, any of the measured parameters o f diabetic control, insulin levels, blood pressure, or lipoprotein cholesterol or triglyceride levels.

In subjects randomized to placebo, L-arginine, atorvastatin or combined therapy, FMD changed by -0.33 ± 2.11%, -0.33 ± 2.49%, 1.75 ± 2.88% and 0.68 ± 2.45% respectively (Figure 5.3.2). Of the prespecified baseline covariates, baseline FMD (p = -0.37, P = 0.003) and duration of diabetes (p = -0.07, P = 0.03) were significantly associated with change in FMD. After allowing for the effect of these, atorvastatin therapy was associated with a significant increase in FMD of 1.26% (95%CI: 0.22 to 2.3; P = 0.018) (Table 5.3.4). L-Arginine was not associated with a significant change

Table 5.3.3 Effect of atorvastatin on cholesterol and triglyceride levels

A to r v a s ta t in P l a c e b o

Baseline 6 weeks

%

change Baseline 6 weeks % change P

No 41 36 42 41

Total cholesterol (mmol/l) 5.00 ± 0.96 3.31 ±0.66 -33.3±9.4 4.73 ± 0.94 4.70 ± 0.97 -0.08 ± 8.8 <0.001

LDL cholesterol (mmol/l) 3.05 ± 0.86 1.55 ±0.48 -48.3 ± 10.0 2.90 ±0.92 2.81 ± 0.92 -9.5 ± 16.6 <0.001

HDL cholesterol (mmol/l) 1.54 ±0.51 1.41 ±0.44 -6.1 ±21.3 1.39 ±0.40 1.42 ±0.39 4.3 ±23.5 0.04

Triglycerides (mmol/l) 0.90 ± 0.39 0.76 ± 0.29 -12.1 ±25.5 1.01 ±0.83 1.08 ± 1.01 3.7 ±32.1 0.02

a\

L-Arginine had no effect on any of the lipid parameters measured, therefore, data shown is only segregated by randomisation to atorvastatin.

in FMD (-0.51%; 95%CI: -1.54 to 0.51; P = 0.32) and in subjects who received L- arginine in addition to atorvastatin there was a trend towards reduction in the benefit of atorvastatin therapy (-1.28% (95%CI: -3.35 to 0.78), P = 0.2) but this was not significantly different from the effect of atorvastatin alone (Table 5.3.4). Other variables including lipoprotein cholesterol levels, plasma glucose level, fructosamine and blood pressure were added to this model together with interactions between variables already in the model but none were found to have a significant effect. None o f the treatment variables or prespecified covariates were significantly associated with change in GTN mediated dilatation (Table 5.3.5) indicating that the improvement in FMD seen in the atorvastatin subjects is likely to reflect enhanced endothelial derived NO bioavailability.

Simple correlation coefficients were used to examine the relation between improvement in FMD and the change in cholesterol levels. There was a borderline significant inverse correlation between improvement in FMD and reduction in total cholesterol (r = -0.21, P = 0.07), but no significant relation with change in HDL (P = 0.2) or LDL cholesterol (P = 0.11) or triglyceride (P = 0.16) levels.

Following atorvastatin, plasma levels of vWF decreased from 1.28 ± 0.50 to 1.10 ± 0.28 (P = 0.03 on ANCOVA), there being no significant change following L-arginine therapy. As with FMD, the effect was greatest in subjects who received atorvastatin alone with a relative diminution of the effect in subjects on combined therapy. This interaction did not, however, reach statistical significance (Figure 5.3.3) Neither intervention had any significant effect on plasma levels of PA Il, or fibrinogen.

There was a borderline significant correlation between the reduction in vWF levels and change in total cholesterol levels (r = 0.23, P = 0.06) but no relation with the improvement in FMD (r = -1.63, P = 0.2).

Table 5.3.4 Determinants of change in flow mediated dilation. Source df Sum of Squares Mean Square F P Atorvastatin 1 28.678 28.678 5.84 0.018 L-Arginine 1 4.942 4.942 1.01 0.319 Interaction 1 7.575 7.575 1.54 0.219 Duration of diabetes 1 33.764 33.764 6.87 0.011 Baseline FMD 1 73.312 73.312 14.92 <0.001 Error 69 338.949 4.912 Total 74 487.448

Term Effect Size 95% Cl

Atorvastatin 1.26 0.22 to 2.30 L-Arginine -0.51 -1.54 to 0.51 Interaction -1.28 -3.35 to 0.78 Duration of diabetes -0.07 0.02 to 0.13 Baseline FMD -0.37 -0.56 to -0.18 Constant 0.37 -0.72 to 1.47

The upper table is the analysis of covariance matrix for change in flow mediated dilation (FMD) over the 6-week duration of the study and the lower table gives the regression coefficients (Effect size). Improvement in FMD was significantly associated with atorvastatin therapy even after taking into account the effect of baseline FMD and duration of diabetes. L-Arginine therapy had no significant benefit on FMD and attenuated the effect of atorvastatin when given in combination.

Table 5.3.5 Determinants of change in GTN mediated dilatation Source df Sum of Squares Mean F Square P Atorvastatin therapy 1 34.6 34.6 0.21 0.648 L-Arginine therapy 1 227.6 227.6 1.39 0.243 Interaction 1 127.6 127.6 0.78 0.381 Error 70 11483.6 164.1 Total 75

Term Effect Size 95% Cl

Atorvastatin -1.35 -7.23 to 4.52

L-Arginine -3.50 -9.43 to 2.43

Interaction 5.22 -6.58 to 17.01

Constant 7.87 -1.88 to 17.62

Neither L-arginine or atorvastatin had any independent dilatation to glyceryl trinitrate. Abbreviations: df = degrees of freedom