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]
|
|
|
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.
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
|
|
|
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 |
Subjects and complications |
Age |
Mean |
Duration of |
Conclusions |
Chiarelli et al. |
115 patients: |
18.1 |
8.0 |
14.4 |
Higher plasma
homocysteine in diabetic patients with microvascular complications |
Wiltshire et al. |
78 patients: |
13.7 |
8.8 |
4.5 |
Plasma homocysteine
lower in diabetic group than control group |
Cotellessa |
112 patients: |
17.6 |
Not quoted |
9.4 |
Plasma homocysteine
significantly lower in children and adolescents with type 1 diabetes |
Pavia et al. |
91 patients: |
11-18 |
9.2 |
54% > 5 |
No difference between
diabetic patients (including those with microaneurysms) and control groups |
Vaccaro et al. |
66 patients: |
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. |
75 patients: |
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. |
79 patients: |
|
|
|
Significantly
increased plasma homocysteine found in patients with proliferate retinopathy
but only in those with concomitant nephropathy |
Cronin et al. |
119 patients: |
22-56 |
6.4-8.7 |
4-30.7 |
Low plasma
homocysteine in males with no complications. |
Robillon et al. |
41 patients: |
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. |
75 patients: |
51±13 |
7.6% |
> 10 |
Higher plasma
homocysteine in diabetic patients compared to controls |
Targher et al.[14] |
60 patients: |
32 years |
6.7% |
14.1 |
Higher plasma
homocysteine in diabetic patients compared to controls |
Table 2. Determinants
of Plasma Homocysteine in Type 1 Diabetes
General factors
Factors specific to
type 1 diabetes
|
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.]