The effects of oral vitamin supplementation on cardiovascular risk facto

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The effects of oral vitamin supplementation

on cardiovascular risk factors

JoAnn Guest

Feb 22, 2004 16:48 PST

 

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The effects of oral vitamin supplementation on cardiovascular risk

factors

 

BY J. V. WOODSIDE, I. S. YOUNG, J. W. G. YARNELL, D. McMASTER, A. E.

EVANS [excerpted from the Proceeding of the Nutrition Society

(1997), 56, 479-488, University of Ulster at Coleraine on 24-28

June, 1996]

 

 

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CHD (Coronary Heart Disease) causes approximately half the deaths

among middle-aged adults in the industrialized world. However, major

accepted risk factors combined can explain only about 50% of heart

disease

(Editorial, 1984). The possible aetiological involvement of novel

risk factors, therefore, is receiving much attention.

 

The present review focuses on two such risk factors:

hyperhomocysteinaemia (elevated homocysteine levels) and LDL

oxidation.

 

Both these factors can be linked with inadequate vitamin intake and,

therefore, may be amenable to nutritional intervention (Selhub et

al. 1993; Jha et al. 1995).

 

HYPERHOMOCYSTEINAEMIA

 

Homocysteine is a Sulfur-containing amino acid which is an

intermediary product in methionine metabolism (Finkelstein, 1990).

 

Recent investigations have focused on the possibility that moderate

elevations may be associated with increased risk of vascular disease

(McCully,

1983). To date, more than twenty clinical studies involving over

2000 patients with cardiovascular disease and a similar number of

controls have shown that patients tend to have higher homocysteine

levels, even though in most cases values are within the accepted

normal range

(Malinow, 1990; Kang et al. 1992; Ueland et al. 1992).

 

Several retrospective and cross-sectional studies have linked

premature vascular disorders including CHD, cerebral and peripheral

vascular disease with elevated homocysteine levels (Malinow, 1990;

Clarke et al. 1991; Malinow et al. 1993; Boushey et al. 1995).

 

In addition, the association between hyperhomocysteinaemia and

cardiovascular disease has been confirmed in several large

prospective studies (Taylor et al. 1991; Stampfer et al. 1992;

Verhoef et al. 1994; Arnesen et al. 1995; Perry et al. 1995) with

only one study showing no association (Alfthan et al.

1994).

 

In the Physicians' Health Study, a total of 14916 US male

physicians aged 40 to 84 years were followed up for 6 years. Men

with homocysteine levels above the 95th percentile (based on

control, distribution) had a three-fold increased risk of myocardial

infarction compared with those in the bottom 90 % (Stampfer et al.

1992).

 

The findings were also statistically compatible with a graded risk

increase across the distribution, a suggestion confirmed by Perry et

al. (1995) in a prospective study of stroke in middle-aged British

men. Similar findings have been reported for myocardial infarction

(Arnesen et al.

1995), carotid-artery thickening (Malinow et al. 1993) and

angiographically-defined coronary artery stenosis (Genest et al.

1990).

 

In addition, Selhub et al. (1995) demonstrated a gradual increase in

the prevalence of carotid artery stenosis with increasing levels of

homocysteine. Meta-analysis by Boushey et al. (1995) showed an

increase in risk of coronary artery disease of about 70 % for each 5

umol/l rise in fasting homocysteine.

 

Several mechanisms are likely to be involved in the induction of

vascular disease by homocysteine, including endothelial cell

desquamation (Harker et al. 1974; Starkebaum & Harlan, 1986),

oxidation of LDL (Heinecke et al. 1987; Parthasarathy, 1987; Blom et

al. 1995), and monocyte adhesion to the vessel wall (Kottke-Marchant

et al. 1990).

 

Additional roles for homocysteine in haemostasis and atherogenesis

have been suggested but not confirmed. Early studies showed that in

hyperhomocysteinaemic patients, platelet turnover was increased

(Harker et al. 1974), but this has not been reproduced (Uhlemann et

al. 1976).

 

Coagulation protein function may be disturbed as indicated by low

factor VII levels (Munnich et al. 1983) and reduced functional anti-

thrombin III activity (Giannini et al. 1975). It has been suggested

that a hypercoagulable state is associated with

hyperhomocysteinaernia since there have been many reports of altered

endothelial cell function,

including enhanced factor V activity (Rodgers & Kane, 1986),

decreased protein C activation (Rodgers & Conn, 1990), diminished

fibrinolysis (Harpel et al. 1992) and increased tissue factor

activity (Rodgers et al. 1993). It must be noted, however, that

these effects are not homocysteine-specific; a variety of free thiol-

group amino acids show similar tendencies (Rees & Rodgers, 1993).

 

Metabolism

 

Homocysteine is formed as a product of the S-adenosyl-L-homocysteine

hydrolase (EC 3.3. 1. 1) reaction which is responsible for the

removal of S-adenosyl homocysteine, a product of Sadenosyl

methionine-dependent transmethylation.

 

Intracellular homocysteine is (1) remethylated to methionine, the

transmethylation pathway; (2) converted to cystathionine, the trans-

sulphuration pathway, or (3) exported from the

cells (Fig. 1). Pathway I is catalysed by the enzyme methionine

synthase (EC 2.1.1.13) which requires cobalamin (vitamin B12) as a

cofactor; the methyl donor is 5-methyltetrahydrofolate. During

pathway 2, the vitamin B6-dependent enzyme cystathionine B-synthase

(EC 4.2.1.22) catalyses the irreversible condensation of

homocysteine with serine to form

cystathionine.

Release of homocysteine into the extracellular medium

represents the third route of cellular homocysteine disposal.

 

Measurement of homocysteine

 

Homocysteine exists in several forms in plasma from healthy subjects

with 70 % of homocysteine from healthy subjects associated with

plasma protein (Ueland et al. 1993). The sum of all homocysteine

species in plasma (free plus protein-bound) is referred to as total

homocysteine (tHcy). To measure total homocysteine, plasma is first

subjected to a reducing agent to cleave the disulphide bonds of

homocystine,

homocysteine mixed disulphide and protein-bound homocysteine. The

plasma is then deproteinized and homocysteine levels measured. The

range for total homocysteine values differs somewhat from one

laboratory to another, but values between 5 and 15 umol/l are

usually considered as normal (Ueland et al. 1993).

 

Homocysteine can be measured in plasma after fasting or after an

oral methionine load. In the meta-analysis of Boushey et al. (1995),

the summary risk estimates based on studies which measured fasting

levels were of similar magnitude to those based on fasting and post-

load; suggesting that both fasting and post-load homocysteine are

equally

strongly related to risk of cardiovascular disease.

 

Causes of hyperhomocysteinaemia

 

Total plasma homocysteine seems to be dependent on age, gender, and

menopausal status (Ueland et al. 1993). Moreover, both environmental

and genetic influences contribute to hyperhomocysteinaemia.

 

Environmental and dietary factors. Cross-sectional and experimental

evidence suggests that mild hyperhomocysteinaemia. may be related to

sub-optimal levels of folic acid, pyridoxine and cyanocobalamin, all

cofactors in homocysteine metabolism (Park & Linkswiler, 1970;

Slavik et al. 1982; Smolin & Benevenga, 1982; Smolin et al. 1983;

Kang et al. 1987; Brattstrom et al. 1988; Lindenbaum et al. 1988;

Stabler et al.

1988; Miller et al. 1992).

 

In a study of vitamin and homocysteine levels

in an elderly population, Selhub et al. (1993) found a strong

inverse correlation between homocysteine and plasma folate, and

weaker inverse correlations between homocysteine and cobalamin and

homocysteine and

pyridoxal-5-phosphate.

 

The authors concluded that elevated homocysteine

levels could, in great part, be due to vitamin status. Ubbink et al.

(1993) produced similar results and confirmed by intervention that a

daily supplementation of folic: acid, pyridoxine and cyanocobalamin

could normalize elevated homocysteine concentrations within 6 weeks.

Ubbink et al. (1994) then looked at the effect of supplementation

with the individual vitamins over 6 weeks.

 

Folic acid supplementation (0.65mg/d) reduced plasma homocysteine

concentrations by 41.7 % and cyanocobalamin (0-4 mg/d) by 14.8 %.

The pyridoxine supplement (10 mg/d) had no significant effect on

homocysteine concentrations. The combination of the three vitamins

reduced circulating homocysteine concentrations by 49.8 % which was

not significantly different from the

reduction achieved by folate supplementation alone. No trial has, as

yet, measured the effectiveness of vitamin supplementation with a

clinical endpoint, i.e. a reduction in cardiovascular events.

 

The question arises as to whether dietary change in B-vitamin intake

can reduce plasma homocysteine, or whether food fortification or

vitamin supplementation is necessary. Selhub et al. (1993) found

that 400ug folate/d was necessary to prevent elevation of

homocysteine and this intake was only attained by 30-40 % of

participants in the Framingham study.

 

Boushey et al. (1995) assessed that if the US population were to

eat two to three more servings of fruit and vegetables daily, this

would lead to a reduction of 4 % in deaths from cardiovascular

disease annually through homocysteine reduction.

 

Cuskelly et al. (1996) looked at the effects of increasing dietary

folate on erythrocyte folate. Erythrocyte folate concentration

increased significantly over 3 months in subjects taking folic acid

supplements or food fortified with folic acid (such that folic acid

intake increased by 400 ug/d).

 

There was no increase in those given food naturally rich in

folate (again to provide an estimated increase in intake of 400

ug/d) and those offered dietary advice only. Their likely

explanation lies in the known higher bioavailability of folic acid

compared with food folates. Further investigation is needed using

larger numbers of

subjects to assess the relative effectiveness of a high-folate diet,

food fortification with folate and folic acid supplementation on

plasma homocysteine.

 

Genetic factors. The classical syndrome homocystinuria defines a

group of inherited disorders characterized by the excretion of large

amounts of homocysteine in the urine.

 

Common clinical signs are mental retardation, ectopic lens,

premature vascular disease and thrombosis

(Ueland et al. 1992; Mudd et al. 1989). The most common cause of

homocystinuria is homozygosity for cystathionine B-synthase

deficiency

which has a prevalence of 1:200000 worldwide, although this varies

between populations (1:60000 in Ireland). Other causes are various

defects of homocysteine remethylation, including homozygosity for

5,10methylenetetrahydrofolate reductase (EC 1.7.99.5; MTHFR)

deficiency and errors of cobalamin metabolism.

 

While heterozygotes for these conditions have elevated plasma

homocysteine, this is a relatively rare explanation for

hyperhomocysteinaemia in population studies.

 

Kang et al. (1988a,b) described a thermolabile mutant of MTHFR that

occurs in neurologically-normal patients. This defect is inherited

recessively and the enzyme appears to have about 50 % of the

activity of normal MTHFR and can result in moderate elevation of

homocysteine levels

in blood (Kang et al. 1991a,b, 1992).

 

Kang et al. (1991b) found the

thermolabile variant to be present in about 5 % of the general

population and 17 % of patients with coronary artery disease.

However, many of those with the thermolabile genotype do not have

the hyperhomocysteinaemic phenotype, and in the Kang et al. (1987)

original study, hyperhomocysteinaemia was associated with low plasma

folate concentrations; folate supplementation normalizing these

levels.

 

Thus, there is thought to be an association between thermolabile

MTHFR status, folate and homocysteine levels. Two recent studies

have confirmed this. Jacques et al. (1996) screened 365 individuals

from the NHCBI Family Heart Study. Among individuals with lower

plasma folate, (< 15 4

nmol/1), homozygous thermolabiles had fasting homocysteine levels

that were 24 % greater than individuals with the normal genotype. A

difference between genotypes was not seen among individuals with

folate levels > 15.4nmol/l. Harmon et al. (1996) looked at 625

working men aged 30-49 years, measuring genotype, plasma

homocysteine, serum folate and vitamin B12. The homozygous

thermolabile genotype was observed in 48, 36

and 23 % of the top 5, 10, and 20% of individuals respectively

ranked by homocysteine levels, compared with a frequency of 11.5 %

in the study population as a whole, establishing that the mutation

is a major determinant of homocysteine levels at the upper end of

the range.

 

Thermolabile homozygotes had a 9.7-fold risk of being in the top 5 %

of the homocysteine distribution, compared with non-thermolabile

homozygotes. Both serum folate and vitamin B12 varied with genotype,

being lowest in the thermolabile homozygotes. These studies are

important in that they indicate that individuals who are homozygous

for thermolabile MTHFR may have a higher folate requirement for

regulation of homocysteine.

 

In conclusion, therefore, there is strong support for homocysteine

as an independent risk factor for atherosclerosis. Further work in

this area should be directed towards developing a better

understanding of the interplay between the nutritional and genetic

factors which contribute to hyperhomocysteinaernia. Intervention

studies with clinical endpoints are also required to assess the

effect of strategies designed to lower plasma homocysteine.

 

SUSCEPTIBILITY OF LDL TO OXIDATION

 

Increased oxidation of LDL is one mechanism which has been proposed

to contribute to the atherogenic potential of homocysteine

(Parthasarathy, 1987), and it is currently believed that oxidation

of LDL is a key early stage in the development of atherosclerosis.

Some reports have suggested the presence of oxidatively-modified LDL

in plasma (Itabe et al. 1996), but most oxidation is believed to

occur in the arterial wall, where LDL may be in a microenvironment

in which the antioxidants which normally prevent lipid peroxidation

can become depleted.

 

The major nutritional

factors which may protect LDL against oxidation include tocopherols,

retinol and the carotenoids, ascorbate and the flavonoids. The fatty

acid composition of the diet is also an important factor determining

the susceptibility of LDL to oxidation, with monounsaturated fatty

acids protecting LDL against oxidation (Parthasarathy et al. 1990).

 

Several mechanisms are likely to contribute to LDL oxidation. The

four major cell types within atherosclerotic lesions are endothelial

cells, smooth muscle cells, macrophages and lymphocytes, each of

which can oxidize LDL.

 

Transition metals are potent promoters of freeradical

formation, and there is some evidence suggesting that free Copper

may be present within atherosclerotic plaques (Smith et al. 1992).

 

Oxidation may proceed more rapidly after the introduction of seeding

hydroperoxides into the LDL particle by lipoxygenase enzymes (Folcik

et al. 1995).

 

Regardless of the mechanisms involved, oxidation of

polyunsaturated fatty acids within LDL leads to the formation of

short-chain aldehydes such as malondialdehyde and 4-hydroxynonenal,

which react with key lysine residues on the apoB molecule (Steinberg

et al. 1989).

 

The modified apoB is no longer recognized by the apoB/E

receptor, but instead binds to the scavenger receptor on macrophages

or smooth muscle cells, leading to unregulated uptake of cholesterol

by these cells and the formation of foam cells.

 

These eventually burst and a fatty streak, the first phase of an

atherosclerotic lesion, results.

 

As well as converting macrophages into cholesterol-laden foam cells,

it is now apparent that oxidized LDL may have other diverse

biological effects (Steinberg et al. 1989). It is chemotactic for

monocytes, and once these have entered the arterial wall and

differentiated into macrophages, oxidized LDL may inhibit their

migratory ability and thus trap them (Daugherty & Rateri, 1993).

 

Oxidized LDL is cytotoxic to various cells, including endothelial

cells, because of the lipid peroxidation products it contains

(Witztum & Steinberg, 1991).

 

Oxidized LDL also inhibits endothelium-derived relaxing factor,

which mediates vaso-relaxation of the coronary arteries in response

to agents such as acetylcholine (Rosenfeld, 1991). There is,

therefore, a potential role for oxidized LDL in altering vasomotor

responses, perhaps contributing to vasospasm in diseased vessels. In

addition, oxidized LDL is

immunogenic; auto-antibodies against various epitopes of oxidized

LDL have been found in human serum, and immunoglobin specific for

epitopes of oxidized LDL can be found in lesions (Libby & Hansson,

1991; Salonen et al. 1992).

 

Minimally-oxidized LDL has been shown to stimulate the

secretion of monocyte chemotactic protein by human aortic,

endothelial and smooth muscle cells in culture, increasing the

binding of monocytes to cultured endothelial cells (Cushing et al.

1990) and in addition can stimulate the expression and secretion of

granulocyte and macrophage colony stimulating factors in human

aortic endothelial cells (Rajavashisth et al. 1990). Oxidized LDL,

therefore, may be able to induce arterial wall cells to produce

chemotactic factors, adhesion

molecules, cytokine and growth factors which have an important role

in the development of the plaque (Witztum, 1993).

 

A substantial body of epidemiological evidence supports the

hypothesis that antioxidant dietary factors which can inhibit LDL

oxidation protect against the development of atherosclerosis. A high

intake of antioxidant vitamins, particularly vitamin E, but also

vitamins A, C and B-carotene, is associated with reduced CHD

mortality (Riemersma et al. 1991). Gey et al. (1991) found a strong

inverse relationship between CHD mortality and dietary vitamin E. In

a study of nurses in the USA, heart disease incidence was over one-

third less in those with high intakes of vitamin

E or b-carotene, and numerous other studies support these findings

(Jha et al. 1995). The evidence for the watersoluble vitamin C is

less strong but there may be a synergistic effect between vitamins C

and E (Gey et al. 1991; Kagan et al. 1992). More recently it has

also been suggested

that dietary flavonoids protect against IHD.

 

Numerous intervention studies designed to test the hypothesis that

increased antioxidant intake will protect against atherosclerosis

are currently in progress.

 

In general, these studies are using supplements

of antioxidant vitamins or other antioxidants, and early results

have not been encouraging. Probucol is a lipid-lowering drug with

potent antioxidant properties which can substantially inhibit the

development of atherosclerosis in animal models (Carew et al. 1987).

The Probucol

Quantitative Regression Swedish Trial tested the ability of probucol

to

inhibit the progression of femoral atherosclerosis in man. After 3

years

of follow-up, the probucol group fared no better than the placebo

group

(Walldius et al. 1994), although this may be because probucol

reduces

the HDL2 subfraction in addition to its antioxidant properties

(Johansson et al. 1995). With respect to antioxidant vitamins, The

Alpha-Tocopherol, Beta-Carotene Cancer Prevention Trial in heavy

smokers

found no reduction in CHD morbidity or mortality during 5-8 years of

follow-up (The Alpha-Tocopherol, Beta-Carotene Cancer Prevention

Study

Group, 1994) and the Beta-carotene and Retinol Efficacy Trial was

ended

early when researchers recognized an elevated risk of death from

lung

cancer in those receiving b-carotene (Omenn et al. 1996), although

again

no beneficial effect on cardiovascular disease was found. The

Physicians

Health Study followed more than 22 000 US male doctors treated with

50 mg B-carotene or placebo every other day for an average of 12

years (Hennekens et al. 1996). The trial was conducted in an

exemplary manner

and its results would appear to rule out any beneficial effect with

such supplementation on cardiovascular disease.

 

The Cambridge Heart

Antioxidant Study found that short-term (up to 2 years)

supplementation with a-tocopherol (268-537 mg/d) reduced CHD

morbidity in patients with a history of cardiovascular disease, but

found no benefit in terms of mortality (Stephens et al. 1996).

 

How should we interpret the discordance between epidemiological data

and the results so far available from clinical trials?

 

It may be that the duration of clinical trials is much too short to

show a benefit, and that antioxidant intake over many years is

required to prevent atherosclerosis.

 

In addition, the complex mixture of antioxidant

micronutrients found in a diet high in fruit and vegetables may be

more effective than large doses of a small number of antioxidant

vitamins.

 

This idea is supported by the dramatic benefit of a `Mediterranean'

type of diet in myocardial infarction survivors (Delorgeril et al.

1994). Certainly, the possibility that pharmacological

supplementation with antioxidant vitamins may be harmful at high

doses cannot be discounted, and recommendations relating to the use

of such supplements must await the results of studies in progress.

 

CONCLUSION

 

It is clear that both hyperhomocysteinaemia and LDL oxidation are

associated with CHD and that each of these is in some way linked

with vitamin deficiency. Research is by no means complete and at

present there is uncertainty as to the effectiveness of vitamin

supplementation

versus increased dietary intake to reduce these cardiovascular risk

factors. Further well-designed trials should lead to effective

public health recommendations.

 

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