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Vitamin B Treatment and Cardiovascular Events in Hyperhomocysteinemic Patients 137

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 In addition to this action, homocysteine produces chemical reactions with thiolcombining groups placed in proteins and many other important molecules. The word

“homocysteinylation” means a putative mechanism through which high total plasma

homocysteine levels exert a toxic effect. Homocysteinylation, imagined as similar to diabetic

protein glycation, occurs just on physiological levels of total plasma homocysteine and

depends on concentration and time of exposure. An interesting hypothesis suggests that

cysteine-rich repeated domains of proteins like fibrillin-1 [49], coagulation factors and low

density lipoprotein receptors are sites at risk of homocysteinylation. End-stage renal disease

patients on maintenance hemodialysis have significantly higher total plasma homocysteine

levels and protein-homocysteinylation values as compared to subjects with normal renal

function. It is known that long-term oral folic acid treatment significantly reduces the

alterations of protein functions. Moreover, another relevant toxic action of homocysteine is

DNA hypomethylation, which also can be reverted by folate therapy [50].

HOMOCYSTEINE, VITAMIN B THERAPY AND

VASCULAR EVENTS

Cardiovascular disease is the most important cause of morbidity and mortality not only in

the general population with normal renal function, but also in the end-stage renal disease

patients on dialysis [51,52]. In Italy, the overall mortality rate of end-stage renal disease

patients on dialysis is about 14% per year and cardiovascular events are responsible for up to

50% [53,54].

Epidemiological investigations and succeeding meta-analysis, related to these studies,

have underlined both that total plasma homocysteine concentrations are responsible for 10%

of the cardiovascular disease, and that the homocysteine-reduction net value of 5 micromoles

per liter lowers 25% of cardiovascular events [55,56].

Recently, both the large observational study Dialysis Outcomes and Practice Pattern

Study (DOPPS) showed [57] an association between the regular use of water-soluble

vitamins and lower overall and cardiovascular mortality rate, and our small single-centre

prospective study [58] displayed that homocysteine-lowering vitamin B therapy decreases

cardiovascular events in end-stage renal disease patients on hemodialysis. On the contrary,

two recent papers [59,60] told us that high, rather than low plasma homocysteine levels are

related to lower mortality rate in end-stage renal disease patients on hemodialysis, suggesting

the hypothesis of an inverse epidemiology for homocysteine. These trials had two important

methodological bias, because first the close direct relationship between plasma homocysteine

levels and serum albumin values may simply reflect a malnutrition with an amino-acid pool

reduction and in these two trials either adjustment for albumin levels was not performed, or

when it was done the reverse epidemiology disappeared. Moreover, in both cases, diabetic

end-stage renal disease patients on dialysis were a significant part of enrolled patients, and

they should be analyzed separately because they showed significantly lower homocysteine

130 Marco Righetti

levels as compared with non-diabetic end-stage renal disease patients on hemodialysis.

Consequently, the issue of reverse epidemiology between plasma homocysteine levels and

cardiovascular mortality in end-stage renal disease patients is not settled [61]. Moreover, our

last paper and above all Suliman M et al. [62] recently observed that the paradoxical reverse

association between high total plasma homocysteine values and reduced mortality rate in endstage renal disease patients on hemodialysis may be attributed to the influence of many

puzzling factors on total plasma homocysteine levels, including inflammatory and wasting

markers. Therefore, these works strongly support the theory that the influence of malnutrition

and inflammation on total plasma homocysteine levels should be taken into consideration

when evaluating homocysteine as a risk factor for cardiovascular morbidity and mortality in

end-stage renal disease patients on hemodialysis.

According to my data, supporting the beneficial effect of homocysteine-lowering folic

acid therapy on cardiovascular events also if vitamin B treatment does not normalize plasma

total homocysteine levels in a large part of treated patients, Yap S et al. [63] showed that

long-term treatment with betaine, vitamin B, and low-methionine diet, is effective in

lowering the potentially life-threatening vascular risk in homocystinuric patients, without

reaching normal plasma homocysteine values. Moreover, the effects of homocysteinelowering vitamin B therapy on cardiovascular events has been evaluated also in the general

population with slight increase of total plasma homocysteine levels. It has been recently

published by Yang Q et al. [64] that, after a food fortification program with folic acid, US

and Canada population showed a highly significant decrease of fatal stroke as compared both

with a similar population before the beginning of this program, and with the contemporary

populations of England and Wales not submitted to a food fortification with folate. The same

differences observed with vascular events were obtained considering the effects of food

fortification on neural tube defects. These data are very impressive; also if the intervention

model is not a randomized controlled study which represents the gold standard, but often it is

difficult to obtain in a perfect way with no methodological imperfections. So, a recent metaanalysis by Bazzano LA [65], with regards to the effect of vitamin B therapy on secondary

prevention of cardiovascular disease in randomized controlled studies, found no significant

benefit or harm of folate treatment on the risk of cardiovascular disease, or all-cause

mortality. In contrast they observed, excluding VISP trial by Toole et al [66], a significant

protective effect of folic acid supplementation on stroke (RR, 0.76; 95% CI, 0.63-0.93), and

consequently, they concluded that several ongoing trials might provide a definitive answer to

this important clinical and public health question. VISP trial, also if shows that vitamin B

therapy is ineffective to lower cardiovascular events rate, had two essential methodological

bias: the absence of a placebo group and the food fortification with folate which lowered

homocysteine values in both patients’ groups with, as result, a low difference for

homocysteine levels in the two groups. Therefore the authors decided to renew their data

analysis [67], showing that, if they choose two well-splitted patients’ subgroups for the

vitamin B status, they observe a significant risk reduction for composite cardiovascular

events in patients with both high vitamin B12 levels and high doses of vitamins. Also in the

Norwegian Vitamin Trial (NORVIT) [68] the homocysteine levels are similar in the treated

and untreated groups, and these latter patients show an unexpected increase of folate values

during the study. Moreover, homocysteine levels were not an inclusion criteria, and therefore

Vitamin B Treatment and Cardiovascular Events in Hyperhomocysteinemic Patients 131

a large part of patients had baseline homocysteine levels in the normal range or slightly

increased. Hope-2 trial [69] was published with NORVIT in the same issue of the New

England Journal of Medicine, and it was characterized by the same methodological defects.

Again, baseline homocysteine levels were not inclusion criteria, a large part of the study

population had not hyperhomocysteinemia and folate deficiency because both they originated

from fortification areas like US and Canada and had previously taken vitamin B therapy

before starting the trial. Also the authors of Hope-2 trial tell us that vitamin B therapy do not

reduce the cardiovascular events rate, but they unexpectedly set aside as irrelevant the lower

rate both of combined cardiovascular events (111 vs 147 cases) and of non-fatal

cerebrovascular events (84 vs 117 cases, RR 0.72, 95% CI, 0.54-0.95, p= 0.02) in the vitamin

B subgroup as compared with control group. I think that it is important to underline that all

these randomized controlled trials, excluding our paper, show a small reduction in

homocysteine levels from baseline that could mean a relatively too short follow-up time and,

thus, a powerlessness to show the protective effect of homocysteine-lowering folate therapy

on cardiovascular disease. Moreover, the greatest effect of vitamin B therapy is clearly

achieved when this treatment is assigned to hyperhomocysteinemic patients, as early as

possible, and for a long time.

Haemodialysis vascular access thrombosis is the most common cause of hospitalisation

among haemodialysis patients. In 1999 an observational prospective study by Shemin D et al

[70] showed that 47 of 84 (56%) haemodialysis patients had at least one access thrombosis

during a 18 months follow-up time, and baseline homocysteine values were directly related to

the development of vascular access thrombosis. They also observed that each 1 micromoles/L

increase in the total plasma homocysteine level was associated with a 4% increase in the risk

of vascular access thrombosis. Similar results were published by Mallamaci et al [71] in an

Italian hemodialysis population, because they detected that baseline total plasma

homocysteine values were an independent predictor of arteriovenous fistula outcome.

The retrospective analysis (personal data) of incident end-stage renal disease patients

submitted to hemodialysis from January 01 until now in Vimercate Hemodialysis Unit point

out no baseline biochemical and clinical variables linked to arterio-venous fistula failure, but

considering the parameters as repeated measurements during follow-up time, I discover that

dialysis patients with vascular access dysfunction have significantly higher homocysteine

levels and lower folate values as compared with events-free patients. Moreover, taking into

consideration the patients treated with folate, I detect a significant lower rate of vascular

access failure that it is not obtained when I consider our hemodialyis patients submitted to

anti-aggregant treatment.

In view of these interesting results, it is important to project randomized controlled

clinical trials evaluating the hypothesis that lowering total plasma homocysteine levels may

reduce the rate of haemodialysis vascular access thrombosis.

CONCLUSION

In the last years it has been observed that vitamin B therapy has other essential biological

properties. Folate supplementation has a basic function in one-carbon transfer, involving the

132 Marco Righetti

methabolic process of homocysteine remethylation to methionine, thereby ensuring Sadenosylmethionine, the primary methyl group donor for most biological methylation

reactions [72]. Aberrant patterns and dysregulation of DNA methylation are mechanistically

related to cancers. Indeed, it has been observed an inverse association between folate status

and the risk of several malignancies, in particular colorectal cancer. Hence, modest doses of

vitamin B supplementation may give protection against serious diseases such as

cardiovascular disease and cancer [73] which represent the most important causes of

mortality in the general population and in end-stage renal disease patients submitted to

dialysis or renal transplantation.

Actually, long-term clinical trials have not shown harm from folate supplementation, and

we can affirm that folate therapy is inexpensive and has not actually apparent serious side

effects.

To summarize, I think that it is right to attend other ongoing randomized clinical trials for

give final statements and especially guidelines; but just nowadays, in view of a safe and

unexpensive therapy, it is important to:

a. check total plasma homocysteine levels both in patients with chronic renal failure,

and in patients with normal renal function but with previous cardiovascular events,

b. treat high total plasma homocysteine levels with vitamin B supplementation,

c. correct unbalanced and unrestricted diets because the “food as medicine” is the first

step to reduce the risk of cardiovascular morbidity and mortality.

Naturally, this last thought is not original, because it was first promoted by Hippocrates

2500 years ago.

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In: Vitamin B: New Research ISBN 978-1-60021-782-1

Editor: Charlyn M. Elliot, pp. 121-137 © 2008 Nova Science Publishers, Inc.

Chapter VII

VITAMIN B TREATMENT AND

CARDIOVASCULAR EVENTS IN

HYPERHOMOCYSTEINEMIC PATIENTS

Marco Righetti∗

Nephrology and Dialysis Unit, Vimercate Hospital, Vimercate, Italy.

ABSTRACT

High total plasma homocysteine levels are detected not only in patients with

homocystinuria, a recessively inherited disease, but also in patients with renal failure,

hypothyroidism, and methyltetrahydrofolate reductase polymorphism. The most

important clinical signs of high plasma homocysteine values are thromboembolic

vascular occlusions of arteries and veins, cerebral impairment, osteoporosis, and

displacement of the lens. Cardiovascular disease is the primary reason of morbidity and

mortality in the general population, and it represents about 50% of the causes of

mortality of the patients with chronic renal failure. Folic acid, vitamin B6 and vitamin

B12, lower hyperhomocysteinemia acting on remethylation and transsulphuration

pathway. Vitamin B treatments don't often normalize plasma homocysteine levels, but

long-term effects of vitamin B therapy are effective in reducing the life-threatening

vascular risk of homocystinuric patients. Hyperhomocysteinemia is detected in patients

with chronic renal failure, and especially in patients with stage 5 of chronic kidney

disease. Clinical observational studies have shown different results about the effects of

high plasma homocysteine levels on cardiovascular disease in dialysis patients. In fact,

cardiovascular mortality has been associated not only with hyperhomocysteinemia, but

also in some studies with hypohomocysteinemia. These contrasting data are probably due

to the strict relationship between homocysteine and malnutrition-inflammation markers.

Dialysis patients are frequently affected by malnutrition-inflammation-atherosclerosis

syndrome, and consequently this severe clinical condition can interfere with

∗

 Correspondence concerning this article should be addressed to Dr. Marco Righetti, U.O.C. di Nefrologia e

Dialisi, Ospedale di Vimercate, Via C. Battisti 23, Vimercate 20059, ITALIA. e-mail: righettim@hotmail.com.

122 Marco Righetti

homocysteine levels. I and my coworkers recently observed in a prospective clinical trial

that hemodialysis patients, submitted to vitamin B treatment, with low homocysteine

levels and high protein catabolic rate show a significantly higher survival rate as

compared with the other three subgroups. Prospective clinical studies, evaluating

homocysteine-lowering vitamin B therapy on cardiovascular events in patients with mild

hyperhomocysteinemia, have recently shown no clinical benefits. These results could be

misleading because a part of patients had normal homocysteine levels, follow-up time

may have been too short, and confounding factors has not been considered. To

summarize, this paper shows the hottest news regarding the effects of homocysteinelowering vitamin B therapy on cardiovascular events, exploring the intriguing puzzle of

homocysteine.

Keywords: homocysteine, folic acid, vitamin B, cardiovascular disease.

INTRODUCTION

About 50 years ago homocysteine’s story begins: just on 1955 Vincent du Vigneaud, an

American scientist born in Chicago on 18th May 1901, won the Nobel Prize in Chemistry. He

is considered homocysteine’s father because his researches focused principally on sulphurcontaining molecules and their metabolism like transmethylation and transsulphuration [1].

Homocystinuria and high plasma homocysteine concentrations were first described in the

60s. In 1962 Carson et al. [2] discovered homocysteine in the urine of subjects with cerebral

impairment and skeletal abnormalities, and then in 1969 McCully [3] observed in a postmortem study a link between homocysteine and vascular disease detecting extensive

atherosclerosis in patients with homocystinuria and high homocysteine levels.

Homocystinuria is a recessively inherited disease due to cystathionine beta-synthase

deficiency. Cystathionine beta-synthase catalyzes the first step in the transsulfuration process,

promoting the condensation of homocysteine with serine to form cystathionine. The

biochemical data of this rare metabolic inborn error are: hyperhomocysteinemia with 10

times higher levels than normal, hypermethioninemia, hypocysteinemia; while the clinical

findings are: thromboembolic vascular occlusion of arteries and veins, cerebral impairment,

osteoporosis, skeletal abnormalities, displacement of the lens.

In the last ten years the scientific interest for homocysteine and vitamin B therapy is

highly increased because of its strict relationship with cardiovascular events. This paper

shows the hottest news regarding the effects of homocysteine-lowering vitamin B therapy on

cardiovascular events, exploring the intriguing puzzle of homocysteine.

HOMOCYSTEINE METABOLISM

Homocysteine is a small, 135 Da, sulfur amino acid. Plasma homocysteine is chiefly

bound to albumin, but it exists also as free, non protein-bound, form. Total plasma

homocysteine includes all homocysteine fractions, both protein-bound and free.

Homocysteine is achieved from methionine’s demethylation, and it is then converted either to

Vitamin B Treatment and Cardiovascular Events in Hyperhomocysteinemic Patients 123

cysteine through the transsulfuration pathway or to methionine through the remethylation

process. In the transsulfuration pathway homocysteine is metabolised to cystathionine in a

reaction, requiring vitamin B6, catalysed by cystathionine beta-synthase; while, in the

remethylation process homocysteine acquires a methyl group either from 5-

methyltetrahydrofolate with a vitamin B12 dependent reaction, or from betaine (Figure 1).

Methionine

Hcy

Betaine

Cystathionine

Cysteine

Cystathionine β-sinthase

Cystathionase

THF

5-MTHF

5,10-MTHFR B12

B6

B6

Figure 1. Homocysteine metabolic pathway.

The human cystathionine beta-synthase gene is on chromosome 21, and therefore

patients with trisomy 21 have greater than normal enzyme activity and as a result lower than

normal total homocysteine [4]. Diabetic patients have decreased homocysteine values

because cystathionine beta-synthase activity is increased by the reduced levels of insulin and

by the high levels of counter regulatory hormones such as glucagon and glucocorticoids [5].

Also the enzyme 5,10-methylene-tetrahydrofolate reductase (MTHFR), which reduces

5,10-methylene-tetrahydrofolate to 5-methyl-tetrahydrofolate, may have a reduced activity

due to genetic mutations, and as a result a specific susceptibility to folic acid insufficiency,

high total plasma homocysteine levels, and an increased risk for cardiovascular events [6].

Total plasma homocysteine levels are related to sex and age: young men usually have

higher homocysteine values than women of the same age, but after fertility period this gender

difference disappears probably because the positive effect of estrogens [7] promptly goes

away with menopause; while the age-related increase of plasma homocysteine levels is

probably linked to the physiologic reduction of renal function. High homocysteine levels are

frequently detected in end-stage renal disease patients. Plasma homocysteine values rise as

renal function declines, and total homocysteine levels fall in patients after renal

transplantation [8], suggesting that renal mechanisms are at least partly responsible for the

increase of homocysteine among individuals with renal impairment [9]. High plasma

124 Marco Righetti

homocysteine levels in patients with renal failure don’t directly depend on impaired renal

excretion because, first, protein-bound form is not filtered and, second, almost all filtered free

fraction is submitted to proximal tubular reabsorption. The most apt hypothesis is the increase

of uremic toxins that may lead to impairment of enzymes related to homocysteine

metabolism. Methionine transmethylation and homocysteine remethylation are decreased in

patients with renal failure compared to healthy subjects, in contrast, whole body

homocysteine transsulfuration appears to be unaffected when corrected for variation in the

B6 vitamin status [10]. Vitamin B11, isolated from spinach leaves and called folate from the

Latin “folium” [11], is the most important determinant of plasma total homocysteine. Folate

therapy increases homocysteine remethylation and methionine transmethylation, and almost

certainly indirectly stimulates cystathionine beta-synthase [12] improving transsulfuration,

but not sufficiently to normalize homocysteine in the major part of end-stage renal disease

patients [13].

Total plasma homocysteine levels depend also on thyroid state: hypothyroidism is

associated with low and hyperthyroidism with high glomerular filtration rate, which in turn is

strictly related to plasma total homocysteine [14]. Therefore total homocysteine is decreased

in hyperthyroidism, and increased in hypothyroidism. Furthermore, in hypothyroidism

hormone replacement therapy normalizes homocysteine levels [15].

Plasma total homocysteine exists essentially as the protein-bound form, with albumin

being the main homocysteine-binding protein, and this is showed by a positive relationship

between plasma total homocysteine and serum albumin in end-stage renal disease patients.

Another important finding in these patients is the positive correlation between plasma total

homocysteine and serum creatinine, even stronger than that seen with serum albumin. This

finding may strengthen a nutritional factor of total homocysteine, but it could also be the

result of the metabolic association between total homocysteine and serum creatinine. In fact,

the formation of creatine, the precursor of creatinine, depends on methyl donation by Sadenosyl-methionine to become S-adenosyl-homocysteine, leading to the formation of

homocysteine [16]. Plasma total homocysteine may be a nutritional marker in maintenance

dialysis patients, and this nutritional feature may explain its reverse association with

mortality rate in some studies [17].

Diabetic patients on dialysis have higher homocysteine levels than diabetic patients with

normal renal function, but lower than dialysis patients with other nephropathies [18].

Many drugs may influence plasma total homocysteine levels. Methotrexate, “classical

antifolate” used in the treatment of cancer, as well as for other conditions such as rheumatoid

arthritis and psoriasis, interrupts the function of folate’s methyl transfer. Treatment protocols

with methotrexate can induce an acute state of folate depletion which may lead to significant

treatment-related toxicity. Both folate and folinic acid reduce methotrexate toxicity, and

decrease methotrexate-induced hyperhomocysteinemia. The efficacy of methotrexate

probably decreases slightly, but the benefit outweighs the risk. Folate supplementation

should, therefore, be routinely prescribed to every patient taking low-dose methotrexate [19].

Phenytoin, phenobarbital and primidone are also associated with high plasma total

homocysteine and low folate levels, whereas valproate does not influence folic acid and

homocysteine [20]. Moreover, both Parkinson’s disease patients treated with L-dopa and

asthma patients treated with theophylline show high homocysteine levels because in the first

Vitamin B Treatment and Cardiovascular Events in Hyperhomocysteinemic Patients 125

ones most likely the breakdown of L-dopa by catechol-O-methyltransferase results in

increased homocysteine formation [21], and in the second ones theophylline, a pyridoxal

kinase antagonist, causes vitamin B6 deficiency, impaired transsulfuration and therefore high

homocysteine levels [22]. Conversely estrogens lower homocysteine levels, but the

mechanism behind this observation is unclear. Estrogen-induced lowering of homocysteine

levels is probably not linked to transmethylation, remethylation, and transsulfuration

pathways, but due to a change in albumin metabolism. Furthermore, it is noteworthy to

remember that the influence of anticalcineurin drugs on homocysteine levels is controversial.

Homocysteine levels are closely related with serum creatinine both in cyclosporine and in

tacrolimus treated patients; the latter ones have lower homocysteine levels because they show

higher creatinine clearance.

Table 1 summarizes the effects of drugs and diseases on homocysteine levels.

Table 1. Drugs and diseases affecting total plasma homocysteine levels

Drugs and diseases homocysteine

Vitamin B6 ↓

Folic acid ↓

Vitamin B12 ↓

N-acetylcysteine ↓

Dialysis ↓

Diabetes ↓

Estrogen ↓

Thyroid hormone ↓

Renal dysfunction ↑

Methotrexate ↑

Trimethoprim ↑

Theophylline ↑

Fibrates ↑

Antiepileptic drugs ↑

Metformin ↑

Omeprazole ↑

Levodopa ↑

Cyclosporin ↑

Smoking ↑

Caffeine ↑

Alcohol ↑

HOMOCYSTEINE-LOWERING THERAPY

Total plasma homocysteine concentrations have a strong inverse correlation with serum

folate values. Folic acid supplementation is an effective therapy to normalize total plasma

homocysteine levels in patients with occlusive vascular disease and without renal failure [23].

126 Marco Righetti

On the contrary, high “pharmacological” doses of folate lower, but rarely normalize total

plasma homocysteine levels in patients with end-stage renal disease. We observed in a longterm randomised controlled trial [24] that about 90% of end-stage renal disease patients on

hemodialysis, treated with folic acid, have total plasma homocysteine levels higher than the

upper normal limit, and that folate treatment with 15 mg per day is not better than 5 mg per

day in lowering total plasma homocysteine levels. Folate supplementation with higher doses,

equal to 30 or 60 mg per day, is not more useful than 15 mg per day in reducing high total

plasma homocysteine levels [25]. Moreover, it has been observed that the supplementation

with folate and betaine does not further reduce total plasma homocysteine values [26],

suggestive of a betaine-dependent remethylation not stimulated by exogenous betaine when

patients are just submitted to folate therapy; and also that the homocysteine-lowering effect

of i.v. folinic acid, oral folinic acid and oral folic acid is similar [27], suggesting that high

total plasma homocysteine levels are not due to abnormal folate metabolism.

We recently detected in a 6-months prospective trial [28] that vitamin B therapy,

including folate 5 mg p.o. per day, vitamin B12 1 mg i.m. per week, and vitamin B6 300 mg

p.o. per day, largely reduces total plasma homocysteine levels, normalizing these values in

more than 70% of end-stage renal disease patient on peritoneal dialysis. We chose to add

high doses of vitamin B12 and vitamin B6 to standard folate therapy because:

1. the remethylation of homocysteine to methionine needs vitamin B12 as enzymatic

cofactor;

2. the folate supplementation reduces the dependency of homocysteine on folate with a

shift in dependency from folate to vitamin B12 [29];

3. the transsulfuration of homocysteine to cystathionine needs vitamin B6 as enzymatic

cofactor; and

4. vitamin B6 deficiency, usually found in dialysis patients, contributes to impaired

transsulfuration.

The literature’s data tell us that there are no known severe side effects concerning folate

therapy; and, furthermore, the upper level of 1 mg per day of folic acid recommended dose

[30] is due to the possible risk of concealing anemia in the case of vitamin B12 deficiency.

Also for vitamin B12 supplementation, there are no known side effects, and apparently

neither upper limits of intake. Vitamin B6 is important, when added to vitamin B12 and

folate, to reduce hyperhomocysteinemia; but single high doses of vitamin B6 are

unsuccessful to lower high total plasma homocysteine levels. Moreover, contrary to folate

and vitamin B12, there is a safe upper limit for long-term vitamin B6 supplementation that is

equal to 50-100 mg per day. Table 2 shows the different doses of vitamin B therapy in the

homocysteine-lowering trials with a long-term follow-up period.

Total plasma homocysteine concentrations of end-stage renal disease patients may be

also improved with dialysis therapy. The standard low-flux bicarbonate dialysis removes

about 30% of the pre-dialysis total plasma homocysteine concentration and, as expected,

homocysteine reduction rate during this type of hemodialysis is lower than that of creatinine,

according to its protein binding. Total plasma homocysteine levels do not rise for at least 8

hours after standard low-flux dialysis in contrast to plasma creatinine concentration [31], and

Vitamin B Treatment and Cardiovascular Events in Hyperhomocysteinemic Patients 127

plasma homocysteine levels have a postdialytic slight decrease, considering patients on high

flux dialysis [32]. This interdialytic homocysteine curve is fitting with the thinking that

dialysis treatment may remove uraemic toxins with inhibitory activities against one or more

enzymes of the remethylation or transsulphuration pathway. The high-flux dialysis membrane

should perform this removal with greater efficiency. High-flux dialysers with high capacity to

eliminate large uraemic substances, but without excessive leakage of useful proteins such as

albumin, show an intradialytic higher homocysteine-lowering rate, about 40% compared to

30% with low-flux membrane, and pre-dialysis total plasma homocysteine values are slightly,

but not significantly, lower in end-stage renal disease patients treated with high-flux

membranes as compared to patients submitted to low flux dialysers during a follow-up time

of 3 months [33]. The high-flux advanced polysulphone dialysers with high clearance of

larger uraemic toxins, but non-albumin-leaking, do not improve homocysteine clearance

compared to high-flux standard polysulphone membranes, confirming that the large part of

uraemic toxins affecting homocysteine metabolism are protein-bound or have a molecular

weight above 15000 Daltons [34]. The super-flux, albumin-leaking, dialysers improve predialysis total plasma homocysteine values as compared to both low and high-flux membranes,

mainly by removing large molecular weight solutes able to affect the homocysteine

metabolism [35,36,37]. Also the hemodiafiltration with endogenous reinfusion and the

internal hemodiafiltration have high homocysteine-lowering power [38] similar to superflux

membranes. End-stage renal disease patients submitted to pre-dilution on-line hemofiltration

[39], nocturnal hemodialysis six or seven nights per week [40], and peritoneal dialysis

[41,28] show a significantly lower pre-dialysis total plasma homocysteine levels as compared

to patients on standard low-flux hemodialysis, because the first removes larger molecular

weight solutes, and the others have a shorter interdialytic period which permits a less

restricted diet. Indeed, total plasma homocysteine concentrations are not efficiently decreased

by peritoneal dialysis because its dialytic removal via the peritoneal membrane is inefficient

owing to its high protein-bound fraction [42] and, therefore, the reason of lower total plasma

homocysteine levels in end-stage renal disease patients on peritoneal dialysis as compared to

patients on standard hemodialysis is in theory due to the continuous treatment which gives

the chance to the patients having a diet with more fruit and vegetable.

Table 2. Vitamin B doses, net changes of homocysteine from baseline levels, and relative

risk for stroke in the long-term homocysteine-lowering trials

Journal First Author B6 Folate B12 Δ homocysteine RR Stroke

Blood Purif Righetti M [58] 5mg -15.1 0.55

JACC Zoungas S [74] 15mg -2.4 0.45

N Engl J Med HOPE-2 Inv. [69] 50mg 2.5mg 1mg -3.2 0.76

N Engl J Med Bǿnaa KH [68] 40mg 0.8mg 0.4mg -3.8 0.91

JAMA Toole JF [66] 25mg 2.5mg 0.4mg -2.1 1.04

End-stage renal disease patients on maintenance haemodialyis, submitted to intravenous

N-acetylcysteine, showed post-dialysis lower plasma homocysteine levels and better pulse

pressure values as compared with untreated haemodialysis patients [43,44]. Scholze A et al.

[43] demonstrated not only that patients submitted to 5 g acetylcysteine in 5% glucose

128 Marco Righetti

solution for 4 hours during a single haemodialysis session had total plasma homocysteine

levels markedly reduced (about 90%, with post-dialysis homocysteine values equal to 2

micromoles/liter) beyond the effects of haemodialysis alone; but also they observed an

improvement of endothelial function. The high homocysteine-lowering effect of intravenous

acetylcysteine is probably due to a quick displacement of homocysteine from protein-binding

sites, allowing an increased rate of homocysteine available for clearance by haemodialysis,

considering its small size. On the contrary, oral administration of acetylcysteine showed only

a 20% of homocysteine-lowering as compared to no homocysteine-reduction in the placebo

group [45]; and, unfortunately, a randomised controlled trial by Tepel M et al. [46] showing a

significant lowering of composite cardiovascular end-points in haemodialysis patients

submitted to acetylcysteine, 600 mg BID orally, did not analyze the effects on plasma

homocysteine.

A recent paper [47] has shown preliminary data concerning the action of mesna, a thiolcontaining drug analogue of taurine used to protect the bladder wall from haematuria and

haemorrhagic cystitis caused by cyclofosfamide and other cancer-fighting drugs, on total

plasma homocysteine levels in hemodialysis patients. The intra-dialytic mesna

supplementation at the dose of 5 mg per Kg caused, lowering homocysteine’s protein-bound

fraction, a higher decrease (about 55%) of total plasma homocysteine levels as compared to

hemodialysis alone (about 35%). Table 3 summarizes homocysteine-lowering treatments.

Table 3. Homocysteine-lowering treatments in end-stage renal disease patients

First Author Rx Result

Righetti M [24] 5mg FA 5mg FA is similar to 15mg, only about 10% of pts with

final normal homocysteine values

Righetti M [28] 5mg FA, 250mg B6,

500mg B12

Multivitamin B therapy is better than FA alone, about

70% of pts with final normal homocysteine values

Righetti M [38] HDF High intra-dialytic homocysteine-lowering rate, about

40-50%, in pts on I-HDF, OL-HDF, HFR; better than

30% in pts on standard thrice weekly HD

Moustapha A [41] PD Homocysteine levels are lower in pts on PD as compared

with pts on thrice weekly HD

Friedman AN [40] Every-day HD Homocysteine levels are lower in pts on every-day HD

as compared with pts on thrice weekly HD

Scholze A [43] N-acetylcysteine IV N-acetylcysteine therapy improves intra-dialytic

homocysteine-reduction rate

HOMOCYSTEINE TOXIC EFFECT

High total plasma homocysteine values cause endothelial damages through several

mechanisms, usually not exclusive [48]. Homocysteine can change the release or activity of

anti-inflammatory, vasoactive agents like adenosine and nitric oxide. High homocysteine

levels are linked to impaired vasodilation and decreased nitric oxide production by

endothelial nitric oxide synthase, due both to arginine transport alterations that reduce

Vitamin B Treatment and Cardiovascular Events in Hyperhomocysteinemic Patients 129

cellular uptake of L-arginine and to the increase of asymmetric dimethylarginine, an

endogenous inhibitor of nitric oxide synthase, with consequent rise of superoxide anion

production.