Plasma total homocysteine in the active stage of ulcerative colitis

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Abstract. Background: Homocysteine, an independent risk factor for thromboembolism, has been recently shown to be elevated in ulcerative colitis (UC).

Blackwell Publishing AsiaMelbourne, AustraliaJGHJournal of Gastroenterology and Hepatology0815 93192006 Blackwell Publishing Asia Pty Ltd200621739743Original Article Homocysteine in ulcerative colitisJ Drzewoski et al.

doi:10.1111/j.1440-1746.2006.04255.x

GASTROENTEROLOGY

Plasma total homocysteine in the active stage of ulcerative colitis Józef Drzewoski,* Anita Gasiorowska,* Ewa Malecka-Panas,* Edward Bald† and Leszek Czupryniak* *Department of Gastroenterology and Metabolic Diseases, Medical University of Lodz and †Department of Environmental Chemistry, University of Lodz, Lodz, Poland

Key words folates, homocysteine, thromboembolism, ulcerative colitis. Accepted for publication 11 August 2004. Correspondence Dr L Czupryniak, Gastroenterology and Metabolic Diseases Department, Barlicki University Hospital No 1, Medical University of Lodz, Kopcinskiego 22, Lodz 90-153, Poland. Email: [email protected]

Abstract Background: Homocysteine, an independent risk factor for thromboembolism, has been recently shown to be elevated in ulcerative colitis (UC). However, its relation to the activity of the disease remain unclear. Methods: Two groups were studied: group consisted of 1–30 patients with UC (17 men, 13 women, mean age 50.3 ± 14.7 years), including 15 patients with active disease. Group 2 (controls) consisted of 21 age-, sex-, bodyweight-matched healthy persons (12 men, nine women, mean age 53.1 ± 12.8 years). Total plasma homocysteine (tHcy) and serum folate and vitamin B12 as well as selected coagulation parameters were assessed. Results: Mean tHcy in UC patients was significantly higher than in healthy controls: 10.8 ± 3.1 mmol/L versus 6.8 ± 2.5 mmol/L (P < 0.001). Patients with active disease had higher tHcy than patients in remission: 11.2 ± 3.5 mmol/L versus 9.0 ± 2.3 mmol/L (P = 0.048). Patients with ≥4 recurrences of the disease had also higher tHcy than the others: 11.5 ± 3.6 mmol/L versus 9.0 ± 2.1 mmol/L (P = 0.035). The tHcy correlated with duration of disease: r = 0.6632 (P < 0.05). Folate and B12 levels were within their reference ranges in all subjects. However, in the patients with active disease the platelet count, fibrinogen and D-dimer were significantly higher than in the patients in remission and the controls. Conclusions: Ulcerative colitis is associated with elevated tHcy concentration, particularly in the active stage, and in more recurrent types of the disease; this elevation does not seem to be prevented by a normal folate status and might have an enhancing effect on the procoagulation blood profile.

Introduction Homocysteine has been recently acknowledged as an independent risk factor for thromboembolism. It is also well known that the prevalence of thromboembolic complications is increased in patients with inflammatory bowel disease, including ulcerative colitis (UC). Studies from the Mayo Clinic reported the incidence to be between 1% and 6%.1 In autopsy studies, however, this rate was markedly higher: up to 39%.2,3 Novacek et al. reported that clinically important thrombotic events, such as pulmonary embolism, peripheral venous thrombosis, and cerebrovascular stroke, are rare in inflammatory bowel disease and occur at a rate of 0.1% per year. Moreover, they are usually accompanied by increased activity of the disease.4 It should be stressed, though, that thromboembolic complications are strongly associated with critical increase in mortality and therefore are an important clinical factor in the course of the disease.5

The etiology and pathogenesis of thromboembolic events in inflammatory bowel disease remain unclear. Several mechanisms leading to a ‘hypercoagulable state’ observed in UC have been suggested: increased platelet activity,6 increased plasma fibrinogen and factor VIII,3 increased thromboplastin generation time,2 impaired fibrinolysis,7 factor V Leiden mutation,8 presence of cardiolipin antibodies,9 and increased lipoprotein (a).10 Also proinflammatory cytokines, such as tumor necrosis factor-α, have been implicated as triggers of coagulation cascade in inflammatory bowel disease.11 However, the results of the published studies have often been conflicting, and thus no clear explanation of increased thrombosis in UC has been formulated so far. Homocysteine, a sulfur-containing amino acid formed solely in the metabolism of methionine, is an established risk factor for venous thromboembolism and cardiovascular disease.12–14 Its blood level is dependent on folate and vitamin B12 intake,15 and it has been repeatedly shown that its level is higher in men than

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women and that it increases linearly with age.16 Numerous studies described the mechanisms through which homocysteine may induce coagulation cascade or injure endothelium.17–20 The reference range for plasma total homocysteine has been accepted according to Kang et al. as 5–15 µmol/L.21 Elevated plasma homocysteine concentration, usually caused by folate deficiency or renal function impairment, has been suggested to play a considerable role in the development of coronary heart disease,22,23 cerebrovascular disease,24 and vascular complications of diabetes.25,26 Recently hyperhomocysteinemia has been reported in inflammatory bowel disease, both UC and Crohn’s disease.27–30 To the best of our knowledge, no study published so far has focused on the relation of the activity of the disease and homocysteine metabolism. We have therefore performed a case–control study aimed at the analysis of homocysteine metabolism in UC patients in the active stage or remission of the disease.

Methods Two groups of subjects were studied. Group 1 consisted of 30 patients with UC (17 men, 13 women, mean age 50.3 ± 14.7 years, range 25–78 years), including 15 patients with active disease. Duration of the disease ranged from 2 to 32 years, mean 9.8 ± 7.3 years. Diagnosis of UC was confirmed in each case by microscopic examination of colon tissue samples. The activity of the disease was assessed according to the disease activity index (DAI), described in detail elsewhere.31 In brief, DAI is based on scoring (0–3 points) of four parameters: stool frequency (range: normal, 0 points; >4 stools per day > normal, 3 points); rectal bleeding (none, 0 points; blood alone passed, 3 points); endoscopic appearance (normal or inactive disease, 0 points; severe, i.e. spontaneous bleeding, ulceration, 3 points); and physician’s assessment of the disease (healthy/remission, 0 points; severe, 3 points). The DAI may yield results ranging from 0 to 12 points, and for the purpose of the present study the disease was regarded active if the DAI was ≥8 points. Eighteen patients were given sulfasalazin (10 with active disease and eight in remission) while nine patients were treated with mesalazin (five with active disease and four in remission). In addition, nine patients in the active stage of the disease were being given oral steroids. Three patients, all in remission, were on no pharmacological treatment. Fourteen patients (nine with active disease and five in remission) had four or more recurrences of the disease. One patient with active disease had a history of deep venous thrombosis. Group 2 was formed by 21 age-, sex-, bodyweight-matched healthy persons (12 men, nine women, mean age 53.1 ± 12.8 years). All the subjects gave informed consent for participation in the study. The study protocol was approved by the local Ethics Review Committee. Blood samples were taken from all study participants in a fasting state between 07.30 and 09.00 hours. The blood was drawn for routine laboratory tests (including blood cell count, glucose, renal and liver tests) as well as for determination of total plasma homocysteine (tHcy, free and protein bound) and serum folate and vitamin B12. Plasma samples for tHcy measurements were frozen in 24°C immediately on centrifugation (800 g for 15 min) and assayed within 4 weeks of collection. The tHcy was measured by high-performance liquid chromatography (HPLC) with Hewlett-Packard 1100 Series system 740

(Waldbronn, Germany), the details of which have been described elsewhere.32 The between-day reproducibility of this method was 5.6% and 2.8% for levels of 6.4 µmol/L and 46.4 µmol/L, respectively. Serum folate and vitamin B12 were measured by microparticle immunoenzymatic (MEIA) method with the use of reagents for IMx system purchased from Abbott Laboratories (Abbott Park, IL, USA). Intra-assay and interassay coefficients of variance for folate and vitamin B12 were 3.8% and 5.1%, and 4.2% and 7.4%, respectively. The reference range for folate was determined as 3.1– 12.4 ng/mL, and for vitamin B12, 223–1132 pg/mL. The following coagulation parameters were also assessed with the use of standard laboratory methods: blood platelet count, fibrinogen, activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer. Statistical analysis was performed using the Statistica for Windows 98 package (version 5.5 PL, StatSoft, Tulsa, OH, USA). Differences between means were analyzed by the Student’s t-test for unpaired data, unless their distribution was not found to be normal with the Kolmogorov and Lillefors tests, in which case the Mann–Whitney U-test was used. Pearson’s correlation coefficient was calculated to describe the relationship between the variables studied. Differences between frequencies were analyzed by the χ2 test or, in cases with subgroup count 0.05). UC, ulcerative colitis.

Table 2 Caogulation parameters UC patients

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Platelet count (10 /L) Fibrinogen (mg/dL) D-dimer (mg/L)

Active stage (n = 15)

Controls Remission (n = 15)

(n = 21)

395 ± 84* 529 ± 137* 0.67 ± 0.35*

267 ± 69 417 ± 106 0.36 ± 0.28

225 ± 60 388 ± 80 0.29 ± 0.1

UC, ulcerative colitis. *P < 0.01 versus the patients in remission and controls

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Discussion The present controlled study confirms the increased prevalence of elevated tHcy in UC patients. Our findings are in accordance with the studies published by Cattaneo et al.,28 Oldenburg et al.,29 Dinca et al.,33 and Danese et al.34 The tHcy values found in the present patients are strikingly similar to those reported in other studies with UC subjects despite the different ethnic and food background of the cohorts.28,29,34 However, the reason for increased prevalence of hyperhomocysteinemia in patients with UC is unclear. The simplest possible explanation is the deficit in folate levels, as described by Cattaneo et al. and Chowers et al.28,35 However, all the patients enrolled in the present study had normal folate and vitamin B12 values so folate deficiency is an unlikely reason for elevated tHcy, as is renal impairment. The indication of the cause of high tHcy in UC is suggested by the fact that the subjects with active disease presented with significantly higher plasma tHcy concentrations and had hyperhomocysteinemia more often than the patients in remission. It may therefore be suggested that active inflammation is associated with tHcy elevation. It has already been shown that pro-inflammatory cytokines may play a role in homocysteine metabolism.11 This hypothesis also explains the increased incidence of thromboembolic complications in the active stage of UC, reported by Novacek et al., because homocysteine is a potent procoagulating factor.36 It also indicates that tHcy levels are not dependent solely on folate status in UC patients, and that some illness-related factor might be involved in the regulation of tHcy levels. This suggestion has already been made by Abbati et al.37 It is worth mentioning at this point that elevated plasma levels of homocysteine may be determined by genes controlling, for example, the expression of homocysteine metabolism-related enzymes.16 Finally, Fredholm et al. postulated that the activation of coagulation cascade in UC patients might be secondary to the inflammation process.38 In their opinion, high tHcy seems to be a good candidate for a link between UC and increased coagulability observed in patients suffering from this disease. In the present study elevated tHcy was associated not only with the active stage of the disease at the moment of blood sampling for tHcy and folate assays, but also with disease duration and number of recurrences. In summary, the patients with more active inflammation over the years were more likely to manifest elevated plasma tHcy levels than those with a milder type of the disease. These findings are not consistent with the results of other authors who found no correlation between tHcy and the activity or duration of the disease.28,29,33,34 This, however, may be explained by the differences in the studied groups. In all these studies patients with UC and Crohn’s disease were pooled together as having inflammatory bowel disease, whereas we have enrolled only patients with a definite diagnosis of UC. Crohn’s disease and UC share some pathophysiological features, but overall they are two distinct clinical entities and we believe that in most cases they should be studied separately. This is of particular importance when activity of a disease is analyzed, because these two types of inflammatory bowel disease have different relapse patterns in terms of clinical and microscopic changes in the intestines. We have also observed that a well-proven positive correlation between age and plasma tHcy39 has been abolished in UC patients. We think that this finding is of considerable clinical importance because it suggests that young persons with UC are subject to tHcy 742

levels typical of older age and thus are exposed to increased risk of not only thromboembolic complications, but also cardiovascular disease. If this observation finds confirmation in other studies, a homocysteine-reducing strategy might be considered beneficial in such patients. It is worth noting that, besides having greater plasma tHcy, the patients with active disease had elevated procaogulation parameters (platelet count, fibrinogen) as well as markers of intravascular thrombosis (D-dimer). These coagulation abnormalities have been repeatedly reported in individuals with UC, in particular in those in the active stage of the disease.3,6,40–42 In the present study these findings clearly suggest that the patients with exacerbation of UC are at generally increased risk of thromboembolic complications, and increased plasma tHcy might be an additional element enhancing this risk. In conclusion, our study indicates that UC is associated with elevated plasma total homocysteine concentration, particularly in the active stage and in more recurrent types of the disease, and that this elevation does not seem to be prevented by a normal folate status. In addition, the patients with the active stage of the disease present with elevated procoagulation parameters, including platelet count and fibrinogen. These findings may offer some explanation for the mechanisms of the increased risk of thromboembolic complications observed in UC.

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