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Thiamine and Benfotiamine dosing of STZ diabetic rats

Animal study groups in this investigation were: normal controls (C), normal controls with high dose thiamine and Benfotiamine therapy (70 mg/kg per day, CT70 and CB70 respectively), diabetic controls (DC) and diabetics given high dose thiamine and Benfotiamine therapy (7 and 70 mg/kg per day, DT7 and DT70, and DB7 and DB70, respectively). At baseline, dietary thiamine exceeded the recommended daily allowance by ca. 6-fold (C), 9-fold (D), 20-fold (DT7), 140-fold (CT70 and DT70), and in thiamine equivalents 20-fold (DB7), and 100-fold (CB70 and DB70).

Body weights of STZ diabetic and control rats

Mean body weights in the control groups increased from 268 and 235 g at baseline to 712 and 607 g at after 24 weeks in thiamine and Benfotiamine dosing studies, respectively; this was not changed significantly by thiamine and Benfotiamine therapy. Mean body weights in the diabetic controls increased from 268 and 243 g at baseline to 351 and 318 g in thiamine and Benfotiamine dosing studies, respectively; similar increases in mean body weight were found for diabetic rats with high dose thiamine and Benfotiamine therapy.

Diabetic state established

The diabetic rats had the characteristics of the diabetic state in both studies: increased plasma glucose concentration and increased glycated hemoglobin HbA1. These increased levels in the diabetic rats were not changed by high dose thiamine and Benfotiamine therapy - Figures 4 and 5.

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Figure 4 - Effect of high dose thiamine (a.) and Benfotiamine (b.) therapy on plasma glucose concentration in STZ diabetic rats and controls

Key: k1 Control; k2 +70 mg/kg thiamine or Benfotiamine; k3 Diabetic control; k4 Diabetic + 7 mg/kg thiamine or Benfotiamine; k5 Diabetic + 70 mg/kg thiamine or Benfotiamine;

5a 5b

Figure 5 - Effect of high dose thiamine (a.) and Benfotiamine (b.) therapy on glycated hemoglobin HbA1 in STZ diabetic rats and controls

Key: key1.gif Control; key2.gif +70 mg/kg thiamine or Benfotiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine or Benfotiamine; key5.gif Diabetic + 70 mg/kg thiamine or Benfotiamine;

Prevention of microalbuminuria and proteinuria by high dose thiamine and Benfotiamine

STZ diabetic rats on insulin maintenance therapy develop microalbuminuria over 24 weeks. Microalbuminuria was evident in the diabetic rats from 6 – 24 weeks, increasing from normoalbuminuria (2.0 – 2.2 mg albumin/24 h) to 12 -17 mg/24 h at 6 weeks with a further progressive increase to 19 - 33 mg/24 h at 24 weeks (P<0.01). Thiamine and Benfotiamine therapy inhibited the development of microalbuminuria by 70 – 80% with no dose-response relationship evident (P<0.01) – Figure 6, a and b. Proteinuria showed similar changes, albeit with excreted amounts of protein approximately 5-fold higher than for intact albumin. Overall, there was no marked difference in potency between thiamine and Benfotiamine.

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Figure 6 - Effect of high dose thiamine (a.) and Benfotiamine (b.) therapy on the development of microalbuminuria in STZ diabetic rats and controls

Key: key1.gif Control; key2.gif +70 mg/kg thiamine or Benfotiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine or Benfotiamine; key5.gif Diabetic + 70 mg/kg thiamine or Benfotiamine;

Hyperfiltration developed in the diabetic controls. This was not prevented by thiamine but it was prevented by Benfotiamine at 6 – 18 weeks. At 24 weeks of Benfotiamine therapy, mild hyperfiltration had developed.

Thiamine status, transketolase activity and expression, and activation of the reductive pentosephosphate pathway

We assessed the effect of high dose thiamine and Benfotiamine therapy on thiamine status of the study group rats by measuring the plasma concentrations of thiamine and thiamine monophosphate (TMP); thiamine pyrophosphate (TPP) was not detectable in blood plasma. We discovered the previously unrecognized characteristic of STZ diabetic rats that they are thiamine deficient – the plasma thiamine concentration was decreased 69% in diabetic rats in the thiamine dosing, with respect to normal controls. This was due to increased urinary excretion of thiamine: urinary thiamine excretion was 0.55 mmol/24 h in controls and 2.28 mmol/24 h in diabetic rats (P<0.01). There was an associated 8-fold increase in the renal clearance of thiamine in STZ diabetic rats, with respect to controls - 0.51 versus 4.03 ml/min (P<0.01). This was prevented by high dose thiamine therapy, producing a dose dependent increase in the plasma concentration of thiamine: 7 mg/kg thiamine normalized and 70 mg/kg thiamine supra-normalized plasma thiamine levels in diabetic rats - Figure 7a. Similar effects were found with high dose Benfotiamine therapy.

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Figure 7 - Plasma thiamine and glomerular transketolase activity in STZ diabetic rats: effect of high dose thiamine therapy

Key: key1.gif Control; key2.gif +70 mg/kg thiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine; key5.gif Diabetic + 70 mg/kg thiamine;

Diabetic rats had decreased TK activity (thiamine study, -29%, P<0.05, P<0.001), which was normalized by thiamine therapy – Figure 7b. There was also a significant but small "thiamine effect" on glomerular TK activity in diabetic rats only: thiamine study 7.4 ± 3.3% (0.05). Decrease in TPP co-factor availability, however, as assessed by the "thiamine effect", did not account for most of the decreased glomerular TK activity in diabetic rats and reversal of this by high dose thiamine and Benfotiamine therapy. Rather, TK expression was decreased in diabetic rats and supra-normalized in thiamine- and Benfotiamine-treated control and diabetic rats. Decreased TK activity was associated with a decreased R5P/GA3P concentration ratio that was increased significantly in both control and diabetic rats by thiamine therapy. The R5P/GA3P concentration ratio (mean ± SEM) in red blood cells was: C 0.91 ± 0.19, CT70 3.05 ± 0.78 (P<0.05 with to normal control), and DC 0.51 ± 0.15, DT7 2.12 ± 0.55 (P<0.05) and DT70 1.71 ± 0.41 (P<0.05 with respect to diabetic control).

Reversal of biochemical dysfunction in hyperglycemia by high dose thiamine and Benfotiamine therapy

Using an assay of in situ PKC activity, we found increased glomerular PKC activity in STZ diabetic rats, with respect to controls (thiamine dosing study 38 ± 3%, Benfotiamine dosing study 54 ± 6%). This was reversed in a dose-dependent manner by thiamine and Benfotiamine therapy – Figure 8, a and b. Both membrane and cytosolic PKC activities were increased in the diabetic controls and increases in both were reversed by high dose thiamine and Benfotiamine.

 8a8b

Figure 8 - Effect of high dose thiamine (a.) and Benfotiamine (b.) therapy on the in situ glomerular protein kinase C activity of STZ diabetic rats

Key: key1.gif Control; key2.gif +70 mg/kg thiamine or Benfotiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine or Benfotiamine; key5.gif Diabetic + 70 mg/kg thiamine or Benfotiamine;

Dicarbonyl compounds such as methylglyoxal, glyoxal and 3-deoxyglucosone are implicated in carbonyl stress and increased formation of AGEs in diabetes, linked to the development of diabetic nephropathy. Methylglyoxal concentration is increased by the degradation of abnormal high concentrations of GA3P and DHAP in cells suffering cytosolic hyperglycemia. High dose thiamine therapy was therefore expected to decrease the plasma concentration of methylglyoxal and this was indeed found. Glyoxal and 3-deoxyglucosone were also increased in the plasma of STZ diabetic rats and surprisingly the increased plasma concentrations of these glycating agents were also decreased by thiamine – Figure 9.

9Figure 9 - Effect of high dose thiamine on the plasma concentrations of α-oxoaldehyde glycating agents in the plasma of STZ diabetic rats

Key: key1.gif Control; key2.gif +70 mg/kg thiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine; key5.gif Diabetic + 70 mg/kg thiamine;

We determined markers of glycation (fructosyl-lysine FL and advanced glycation endproducts AGEs in glomerular protein) and oxidative stress (plasma protein thiols) in STZ diabetic rats with high dose thiamine and Benfotiamine therapy. The methylglyoxal-derived AGEs, MG-H1 and CEL, were increased ca. 2-fold in glomerular protein of diabetic rats and normalized in a dose-dependent manner by thiamine and Benfotiamine – Figure 10, a and b. There were smaller increases in CML in diabetic controls of borderline significance (thiamine study - 0.50 ± 0.19 versus 0.27 ± 0.11 mmol/mol lys; Benfotiamine study – 0.42 ± 0.17 versus 0.26 ± 0.12 mmol/mol lys; P<0.05). These increases were partially reversed by thiamine and Benfotiamine therapy. Glomerular protein FL was increased in diabetic controls but was not decreased by thiamine and Benfotiamine therapy.

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Figure 10 - Effect of high dose thiamine on the glomerular protein concentrations of the advanced glycation endproducts MG-H1 (a.) and CEL (b.)

Key: key1.gif Control; key2.gif +70 mg/kg thiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine; key5.gif Diabetic + 70 mg/kg thiamine;

The concentration of plasma thiols was measured as a marker of oxidative stress. Plasma protein thiols were decreased in STZ diabetic rats by 30% of control levels in both thiamine and Benfotiamine dosing studies. Benfotiamine therapy (50 mg/kg) reversed this decrease significantly but not thiamine (7 and 70 mg/kg) and Benfotiamine (7 mg/kg) did not – Figure 11, a and b.

 11a11b

Figure 11 - Effect of high dose thiamine (a.) and Benfotiamine (b.) therapy on plasma thiol concentration of STZ diabetic rats

Key: key1.gif Control; key2.gif +70 mg/kg thiamine or Benfotiamine; key3.gif Diabetic control; key4.gif Diabetic + 7 mg/kg thiamine or Benfotiamine; key5.gif Diabetic + 70 mg/kg thiamine or Benfotiamine;

Next page: Discussion and Concluding Remarks


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