Homocysteine, Endothelial Dysfunction and Oxidative Stress in Type 1 Diabetes Mellitus

Posted 12/01/2003

Fiona Wotherspoon, David W Laight, Kenneth M Shaw, Michael H Cummings

Abstract and Introduction

Abstract

Type 1 diabetes is associated with an increased risk of cardiovascular disease, which cannot be fully explained by traditional risk factors. Elevated plasma homocysteine is an independent risk factor for macrovascular disease in the general population. This review examines the evidence for hyperhomocysteinaemia in patients with type 1 diabetes and describes the mechanisms that may lead to increased macrovascular susceptibility.

While reports of plasma homocysteine levels in type 1 diabetes are inconsistent, increased plasma homocysteine levels have been found in subgroups of patients with microalbuminuria, nephropathy and macrovascular disease. Although a direct causal relationship between plasma homocysteine and atherosclerosis remains to be proven, potential mechanisms of vascular damage by homocysteine include endothelial dysfunction linked to increased oxidative stress. This could contribute to the association between hyperhomocysteinaemia and macrovascular disease in type 1 diabetes.

Introduction

Hyperhomocysteinaemia is an independent risk factor for atherosclerosis, including cardiovascular (CV) disease, cerebrovascular disease and peripheral vascular disease in the general population.[1] Patients with type 1 diabetes have a two-to four-fold increased risk of vascular disease and vasculopathy is the principal cause of death in these patients.[2] The risk is higher in diabetic patients with incipient and established nephropathy.[3] Mild hyperhomocysteinaemia has been observed in type 1 diabetic patients with microalbuminuria and nephropathy and may explain the increased risk of vascular disease in this high-risk population.[4- 7]

Homocysteine Metabolism

Homocysteine is a sulphur-containing amino acid formed from the metabolism of methionine (figure 1). Dietary methionine is transformed to S-adenosyl-methionine (SAM), a universal methyl donor. S-adenosyl-homocysteine (SAH) a by-product of this reaction is then hydrolysed to homocysteine. The plasma concentration of homocysteine depends on two metabolic pathways, trans-sulphuration and remethylation. In trans-sulphuration homocysteine condenses with serine to form cystathione. This is catalysed by cystathione beta-synthase and requires vitamin B6 as a co-factor. Cystathione is then hydrolysed to cysteine, catalysed by γ-cystathionase. In remethylation homocysteine acquires a methyl group from 5-methyltetrahydrofolate to form methionine. Remethylation is catalysed by methionine synthase and 5,10-methylenetetrahydrofolate reductase and is dependent upon both folate and vitamin B12 as co-factors. In a person eating a high protein diet 50% of homocysteine metabolism will be via trans-sulphuration and 50% via remethylation. One to two per cent of total homocysteine circulates freely in the blood in the reduced form. Seventy to ninety per cent is protein-bound and the remainder circulates as disulphides, homocysteine and the mixed disulphide homocysteine-cysteine.[8] Techniques for measuring plasma total homocysteine (free and protein-bound) include high performance liquid-chromatography, immunoassay and gas chromatography-mass spectrometry.[9] Normal values after an overnight fast lie between 5 and 15 mmol/L.[8]

Click to zoom

Figure 1. (click image to zoom) The metabolism of homocysteine

 

 

Hyperhomocysteinaemia and Cardiovascular Disease

The homocysteine theory of atherosclerosis was first suggested by McCully in 1969, following his observation that children with homocysteinuria and markedly elevated plasma homocysteine levels (> 100 µmol/L) had severe premature arterial disease.[10]

Since then many clinical and epidemiological studies have demonstrated that a mild or moderate increase in plasma homo-cysteine is a risk factor for vascular disease. Plasma homocysteine is known to be associated with other conventional risk factors for vascular disease, however, the association between plasma homo-cysteine and vasculopathy is independent of these other risk factors.[11] This association appears to be dose-dependent and has an additive effect on the risk of all types of vascular disease in smokers and subjects with hypertension.[11] A meta-analysis of studies investigating homocysteine levels and vascular risk concluded that an increase of 5 µmol/L of plasma homocysteine has an equivalent impact on CV risk to an increase of 0.5 mmol/L in total cholesterol.[12]

In a cross-sectional study of 75 patients with type 1 diabetes, macroangiopathy was significantly more common in patients with hyperhomocysteinaemia, having excluded patients with other vascular risk factors.[7] Another study of patients with type 1 diabetes and type 2 diabetes found that plasma homocysteine levels were significantly related to the presence of coronary artery disease and stroke.[13] Hyperhomocysteinaemia is also associated with other traditional cardiovascular risk factors including smoking[14] and hypertension[15] in type 1 diabetes. There are no prospective longitudinal studies of hyperhomocysteinaemia and the development of CV disease in the diabetic population.

 

Plasma Homocysteine Levels in Type 1 Diabetes

Moderate hyperhomocysteinaemia (15-30 µmol/l) has been observed in some studies of patients with type 1 diabetes, although the findings are inconsistent. Adolescent patients with no microvascular complications have lower[16- 18] or similar[19] homocysteine levels compared with non-diabetic controls. Studies in adult patients have demonstrated both similar,[4- 6] lower[20,21] adn higher[6,7,14] plasma homocysteine levels compared with non-diabetic controls (table 1). The homocysteine levels are independent of vitamin status and reflect the heterogeneous nature of the patients studied, including patients with poor glycaemic control, variable duration of diabetes and a variety of microvascular and macrovascular complications. It would appear that certain subgroups are more likely to be associated with hyperhomocysteinaemia (table 2).

 

Determinants of Hyperhomocysteinaemia in Type 1 Diabetes

General factors

Genetic factors. The most common genetic cause of elevated homocysteine in the general population is the C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene.[22] There is substantial variability in the prevalence of this mutation among different ethnic groups with a prevalence of 0-2% in Africans, 12% in Whites and 20% in Asians. TT homozygotes have homocysteine levels 25% higher than those with the wild type CC genotype resulting in reduced enzyme activity and a need for increased dietary folate to maintain adequate enzyme function.[8]

Until recently it has been unclear whether people with the MTHFR mutation have an increased risk of CV disease, however a recent meta-analysis has shown a significantly higher risk of ischaemic heart disease in people with the MTHFR mutation.[23]

In a study of 354 patients with type 1 diabetes the C677T mutation did not significantly affect plasma homocysteine levels and was not associated with an increase of vascular disease,[13] although the MTHFR polymorphism has been observed more frequently in patients with type 1 diabetes and nephropathy compared with those without nephropathy.

Heterozygotes for cystathione beta-synthase deficiency have either normal or mildly elevated fasting homocysteine levels but commonly have abnormally high homocysteine levels following an oral methionine load (100 mg/kg).[1] The incidence of heterozygotes for cystathione beta-synthase deficiency in the general population is less than 1%[8] and has not been studied in type 1 diabetes. Studies have not confirmed an increased risk of CV disease in non-diabetic heterozygotes for the cystathione beta-synthase mutation.[25]

Nutrition. A deficiency in any enzyme or co-factor required for the metabolism of homocysteine can lead to an accumulation of plasma homocysteine. Folate and vitamin B12 deficiency lead to reduced remethylation of homocysteine to methionine and increased plasma homocysteine levels are seen in patients with nutritional deficiencies of folate and B12.[1,25]

B12 and folate deficiency are more common in patients with type 1 diabetes due to the higher incidence of pernicious anaemia[26] and coeliac disease[27] observed in these patients. In the absence of these conditions there is no evidence that B12 and folate deficiency are more common in type 1 diabetes per se. In most studies of plasma homocysteine levels in type 1 diabetes serum B12 and folate levels were in the normal range.[4,5,16,17,19-21,28]

Riboflavin (vitamin B2) is a co-factor for MTHFR and riboflavin deficiency is associated with increased plasma homocysteine levels, particularly in people who have both low folate status and who are homozygous for the MTHFR C677T mutation.[29]

In vitamin B6 deficiency the trans-sulphuration pathway is only mildly impaired and most studies have found that vitamin B6 deficiency is associated with normal fasting homocysteine but elevation of homocysteine after oral methionine loading.[30] There is no evidence of riboflavin or vitamin B6 deficiency in patients with type 1 diabetes. Nutritional deficiencies leading to mild hyperhomocysteinaemia are more common in elderly patients and vegans.[31]

Gene-nutrient interactions. The influence of the MTHFR C677T mutation in determining plasma homocysteine levels is related to the nutritional status of the individual. Homozygotes for the mutation have reduced enzyme activity and elevated plasma homocysteine levels in the presence of folate deficiency. Additional dietary folate supplementation will maintain adequate enzyme function and normalise plasma homo-cysteine levels. Similarly, riboflavin deficiency affects homocysteine metabolism in people with both the MTHFR mutation and low folate status.[29]

Other factors. Other determinants of plasma homocysteine levels include increasing age, male gender and renal failure (see below).

Factors Related to Type 1 Diabetes

Age of onset of diabetes and glycaemic control. In a study of 50 patients with type 1 diabetes those with the earliest age of onset of diabetes (14 years vs. 22 years) and those with poor glycaemic control (HbA1C 8.6% vs. 7.4%) were the most likely to have a rapid increase in their plasma homocysteine levels over a five-year period.[32] This was independent of ageing, duration of diabetes serum creatinine and urinary albumin but was associated with a significant decrease in serum folate levels from 427 nmol/L to 317 nmol/L after five years. The authors suggest that the increase in serum folate levels leads to the increase in plasma homocysteine but the reasons for the lower folate levels in this group of patients are not clear.[32] The association between plasma homocysteine and HbA1C is supported by one other study[16] but is not found in all studies.[4,7,17,19}

Renal function. Renal hyperfiltration. Younger patients and those with no diabetic complications have significantly lower homocysteine levels than non-diabetic controls (p<0.01).17,18,21] The proposed mechanism for this is renal hyperper-fusion. Renal metabolism of homocysteine accounts for a large fraction of total renal clearance of homocysteine. After filtering at the glomerulus, homocysteine is almost completely reabsorbed in the renal tubules and degraded in the renal parenchyma via transmethylation and trans-sulphuration.[33] In the early stages of type 1 diabetes and in those with normoalbuminuria, glomerular filtration rate (GFR) is increased due to hyperfiltration. Wollesen et al. showed that in type 1 and type 2 diabetic patients without nephropathy GFR is a strong determinant of plasma homocysteine concentration independent of age, serum creatinine and serum vitamins.[28] Therefore diabetic patients with relative hyperfiltration and normal serum creatinine have lower than normal plasma homocysteine concentrations.[28]

Diabetic nephropathy. In contrast, hyperhomocysteinaemia is well described in patients with type 1 diabetes and established nephropathy. In these studies homocysteine concentrations are positively correlated with serum creatinine.[5-7,34] Once diabetic patients develop persistent proteinuria there is a progressive linear decline in GFR of about 11 ml/minute/year. It is likely that the increased plasma homocysteine levels seen in diabetic nephropathy are due to this declining GFR and subsequent impaired renal metabolism of homocysteine, however, GFR was not measured in most studies.

Studies of diabetic patients with microalbuminuria and normal serum creatinine have demonstrated mild hyperhomocysteinaemia4,5,7,16,34] but the mechanism of this has not been fully examined. Patients who develop microalbuminuria may have increased, normal or reduced GFR as the microalbuminuria progresses. Some studies report a calculated GFR, however this may be misleading as the Cockcroft-Gault formula can overestimate the measured GFR in patients with type 1 diabetes.[35]

In three studies hyperhomocysteinaemia was positively correlated with albumin excretion rate.[5,7,16] With increasing urinary excretion of albumin, typical structural changes are seen in the glomerulus, including mesangial expansion, thickening of the basement membrane and glomerulosclerosis which could potentially interfere with renal metabolism of homocysteine.

There is little evidence to support a causative role of homo-cysteine in the development of diabetic microvascular disease, as hyperhomocysteinaemia is not seen in patients with diabetic retinopathy without nephropathy.[36]

Non-diabetic patients with chronic renal failure have markedly elevated plasma homocysteine levels (3-5 times normal)[25] and have a high risk of premature vascular disease. Prospective studies confirm that hyperhomocysteinaemia is an independent risk factor for cardiovascular morbidity and mortality in end-stage renal disease.[37] Therefore, hyperhomocysteinaemia in diabetic patients with both incipient and clinical nephropathy may partly contribute to the increased risk of vascular disease in this group of patients.

 

Potential Mechanisms of Homocysteine-Induced Vascular Damage in Type 1 Diabetes

Endothelial Dysfunction

Endothelial dysfunction is an early manifestation of atherosclerosis in patients with type 1 diabetes[38] and may be caused by hyperhomocysteinaemia. In healthy subjects endothelial dysfunction has been demonstrated following an acute increase in plasma homocysteine after an oral methionine load.[39] Plasma homocysteine rose by 2-3-fold from a fasting baseline level of 13-15 µmol/L. These levels are similar to those associated with an increased risk of macrovascular disease in the general population and seen in patients with type 1 diabetes and macrovascular dis-ease. Impaired endothelial function is also seen with physiological increases in plasma homocysteine (2-3 µmol/L) following a methionine load[40] even in subjects in whom plasma homocysteine did not rise above the upper limit of normal (15 µmol/L). This suggests that there may be an incremental deleterious effect of homocysteine on vascular function even with low circulating levels of plasma homocysteine. It could be argued that the methionine load used in these studies may adversely affect endothelial function, however endothelial dysfunction has also been demonstrated in healthy subjects with mild-to-moderate fasting hyperhomocysteinaemia (15-35 µmol/L).[41] There are currently no studies of endothelial function in type 1 diabetic patients with hyperhomocysteinaemia although endothelial dysfunction is likely in hyperhomocysteinaemic diabetic patients.

Lowering Plasma Homocysteine With Folic Acid

Oral folic acid supplementation reduces plasma homocysteine by 25% and the addition of vitamin B12 lowers plasma homocysteine by a further 7%.[42] The evidence that plasma homocysteine causes endothelial dysfunction is further strengthened by studies demonstrating improved endothelial function following treatment with folic acid and vitamin B12. Endothelial dysfunction has been reversed following folic acid and vitamin B12 in healthy subjects with hyperhomocysteinaemia[41,43] and in non-diabetic patients with established coronary artery disease.[44] However, folic acid may also have effects on the endothelium independent of lowering plasma homocysteine. Improved endothelial function has been demonstrated in healthy volunteers with methionine-induced hyperhomocysteinaemia following a single high dose of folic acid with no reduction in plasma homocysteine [45] and following oral folic acid for six weeks independent of homocysteine reduction in non-diabetic patients with coronary artery disease.[46] There are no studies of the effects of folic acid on endothelial dysfunction in patients with type 1 diabetes, and the effects of folic acid supplementation on clinical CV events are not yet known.

Oxidative Stress

The adverse effects of homocysteine on endothelial function may be mediated by reduced production and bioavailability of nitric oxide due to oxidant stress.[47] Hyperhomocysteinaemia could cause oxidant stress via a number of mechanisms (figure 2). In vitro studies using cultured endothelial cells have demonstrated auto-oxidation of homocysteine to form reactive oxygen species,48 including superoxide anion and hydrogen peroxide, increased lipid peroxidation[49] and impaired production of the antioxidant glutathione peroxidase.[50] The plasma homocysteine levels used in these studies are much higher than those found in vivo and these findings need to be clarified by in vivo studies. A clinical study involving patients with inherited defects of homocysteine metabolism found a significant increase in plasma glutathione peroxidase activity and a non-significant increase in red blood cell super-oxide dismutase activity in those with hyperhomocysteinaemia.[51] This may represent up-regulation of antioxidant activity in response to a pro-oxidant insult caused by homocysteine-mediated vascular damage. However, the relevance of these studies to patients with type 1 diabetes is not clear.

Click to zoom

Figure 2. (click image to zoom) Potential mechanisms of homocysteine-induced oxidant stress

In patients with diabetes there is in vitro evidence of reduced platelet nitric oxide synthase (NOS) activity.[52] Incubation of platelet-rich plasma with homocysteine leads to a further reduction in platelet nitric oxide production in patients with type 1 diabetes compared to healthy controls.[53] This reduced platelet-derived NOS leads to increased platelet activation and aggregation and contributes to reduced nitric oxide bioavailability, which provides an additional potential mechanism for the atherogenic action of homocysteine in diabetic patients.

Antioxidants

Endothelial dysfunction in healthy subjects with methionine-induced hyperhomocysteinaemia can be prevented by pre-treatment with the antioxidant vitamins C and E.39,54,55] Vitamin C has also been shown to correct endothelial dysfunction in patients with genetic homocysteinuria.[56] These findings support the hypothesis that the adverse effects of homocysteine on vascular function are mediated through oxidant stress.

 

Conclusions

The relationship between homocysteine and CV disease in subgroups of patients with type 1 diabetes requires further detailed investigation. Data are required to establish hyperhomocysteinaemia as a clinically significant modifiable cardiovascular risk factor and to determine cellular mechanisms of cause and effect.

Reprint Address

Correspondence to: Dr Fiona Wotherspoon Academic Department of Diabetes and Endocrinology, Queen Alexandra Hospital, Southwick Park Road, Cosham, Portsmouth, PO6 3LY, UK. Tel: +44 (0)23 9228 6260; Fax: +44 (0)23 9228 6791 E-mail: Fiona.Wotherspoon@porthosp.nhs.uk

Abbreviation Notes

CV, cardiovascular; GFR, glomerular filtration rate; NOS, nitric oxide synthase; SAH, S-adenosyl-homocysteine; SAM, S-adenosyl-methionine

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Tables for:

Homocysteine, Endothelial Dysfunction and Oxidative Stress in Type 1 Diabetes Mellitus

[Br J Diabetes Vasc Dis 3(5):334-340, 2003. © 2003 Sherborne Gibbs Ltd.]


Table 1. Studies of Plasma Homocysteine Levels in Type 1 Diabetes

 

Author
and year

Subjects and complications
of diabetes

Age
(years)

Mean
HbA
1C(%)

Duration of
diabetes (years)

Conclusions

Chiarelli et al.
2000
[16]

115 patients:
53% microvascular complications
47% no complications
63 non-diabetic controls

18.1

8.0

14.4

Higher plasma homocysteine in diabetic patients with microvascular complications

Wiltshire et al.
2001
[17]

78 patients:
no complications
59 non-diabetic controls

13.7

8.8

4.5

Plasma homocysteine lower in diabetic group than control group

Cotellessa
et al. 2001
[18]

112 patients:
123 non-diabetic controls

17.6

Not quoted

9.4

Plasma homocysteine significantly lower in children and adolescents with type 1 diabetes

Pavia et al.
2000
[19]

91 patients:
8% microaneurysms
no other complications
90 non-diabetic controls

11-18

9.2

54% > 5

No difference between diabetic patients (including those with microaneurysms) and control groups

Vaccaro et al.
2000
[4]

66 patients:
21% microalbuminuria
69% normoalbuminuria
46% no retinopathy
30% background retinopathy
24% proliferate retinopathy
44 non-diabetic controls

43.3±6.8

8.0

17

Plasma homocysteine levels higher in those with microalbuminuria compared with normoalbuminuria and in those with proliferate retinopathy compared with no or background retinopathy

Chico et al.
1998
[5]

75 patients:
37.3% retinopathy
18.7% nephropathy
5.3% macroangiopathy
56 non-diabetic controls

33±12

7.8±2

12.1±11

No difference between type 1 diabetic patients and controls as a whole but plasma homocysteine higher in those with nephropathy and increases in relation to presence and severity of nephropathy

Hultberg et al.
1991
[6]

79 patients:
35% no retinopathy
18% progressive retinopathy
57% proliferate retinopathy
46 non-diabetic controls

 
45±8.6
44.2±7.8
40.6±8.1

 
7.7±1.3
8.9±1.4
8.5±1.4

 
28.8±9.6
26.1±8.8
29.9±8.3

Significantly increased plasma homocysteine found in patients with proliferate retinopathy but only in those with concomitant nephropathy

Cronin et al.
1998
[20]

119 patients:
15% microalbuminuria
34% background retinopathy
10% proliferate retinopathy
45% neuropathy
51 non-diabetic controls

22-56

6.4-8.7

4-30.7

Low plasma homocysteine in males with no complications.
Otherwise no difference between diabetic and control groups

Robillon et al.
1994
[21]

41 patients:
22% microalbuminuria
no neuropathy or retinopathy
40 non-diabetic controls

34.8±12

10.4

10.7±11.1

Plasma homocysteine low in diabetic patients including those with microalbuminuria compared to controls

Hofmann et al.
1998
[7]

75 patients:
48% nephropathy
57% neuropathy
41% macroangiopathy
40 non-diabetic controls

51±13

7.6%

> 10

Higher plasma homocysteine in diabetic patients compared to controls
Hyperhomocysteinaemia in diabetics more common in those with micro-and macrovascular disease

Targher et al.[14]

60 patients:
38% retinopathy
20% microalbuminuria
30 non-diabetic controls

32 years

6.7%

14.1

Higher plasma homocysteine in diabetic patients compared to controls
Higher plasma homocysteine in smokers compared with non-smokers

 

Table 2. Determinants of Plasma Homocysteine in Type 1 Diabetes

 

General factors

  • Genetic
  • Nutrition
  • Increasing age
  • Male gender
  • Renal failure

Factors specific to type 1 diabetes

  • Lower age of onset of diabetes
  • Poor glycaemic control
  • Renal hyperfiltration
  • Diabetic nephropathy



 

References for:

Homocysteine, Endothelial Dysfunction and Oxidative Stress in Type 1 Diabetes Mellitus

[Br J Diabetes Vasc Dis 3(5):334-340, 2003. © 2003 Sherborne Gibbs Ltd.]


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