Giuseppina Mazzola

Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease

Homocysteine, a thiol-containing amino acid, is the branch point between the transsulfuration and the transmethylation pathways of methionine [1]. In the last decade, investigators have studied the role of homocysteine metabolism in the pathogenesis of occlusive vascular disease. Homocystinuria, a disorder caused by homozygous cystathionine -synthase (E.C. 4.2.1.22) deficiency or defects in the vitamin B12 or folate-dependent remethylation of homocysteine to methionine and characterized by fasting plasma homocysteine levels of 200 mol/L or greater, is frequently associated with severe vascular disease during infancy or childhood. More than half of homocystinuric patients have a first thromboembolic event before the age of 30 years, and premature atherosclerosis is a common development [2]. Although the relation of hyperhomocysteinemia to the occurrence of vascular disorders is still unclear, it is of increasing interest that less severe enzymatic abnormalities of the methionine metabolic pathway may predispose to the development of premature vascular disease. Moderate hyperhomocysteinemia, which is characterized by elevations of fasting plasma homocysteine levels as high as 100 mol/L or by abnormally high levels of plasma homocysteine after methionine load [3], has been observed with increasing frequency in young patients with arterial occlusive conditions such as myocardial infarction and cerebrovascular and peripheral occlusive diseases [4-7]. However, although venous thromboembolism accounts for 50% of the vascular complications of homocystinuria, few data are available on the relation between moderate hyperhomocysteinemia and venous thromboembolic disease [8, 9]. The term thrombophilia refers to an increased tendency to thrombosis sustained by an ongoing stimulus to thrombogenesis or by a defect of the natural anticoagulant or fibrinolytic mechanisms [10]. Genetic factors play an important role in thrombophilia because thrombosis is often familial or is associated with congenital deficiencies of the protein C anticoagulant pathway, antithrombin III, heparin cofactor II, or plasminogen. The main clinical features of patients with inherited thrombophilia are recurrent thrombosis, thrombosis at a young age, idiopathic thrombosis, thrombosis after trivial provocation, and thrombosis in an unusual site [10]. The aim of our study was to evaluate the prevalence of moderate hyperhomocysteinemia and of established disorders of inherited thrombophilia in a series of consecutive patients with early-onset venous or arterial occlusive disease. Methods Patients We studied consecutive unrelated patients referred to our coagulation service from November 1992 to October 1994. Patients were enrolled in the study if they were younger than 45 years at the time of the occlusive event (n = 142) or if they had thrombosis at unusual sites (central retinal vein or artery, cerebral veins), provided that local risk factors were absent. Exclusion criteria were the presence of overt cancer, liver disease, acquired coagulation abnormalities (for example, lupus anticoagulants, anti-protein S antibodies, and inhibitors of fibrin polymerization), or risk factors known to disturb methionine metabolism (diabetes, hypertension, hyperlipidemia and renal failure). Thirty-two patients referred during the study period were excluded on the basis of these criteria. At the first study visit, patients were asked [by means of a standard questionnaire] about their personal and family history of thrombosis and whether they had predisposing factors for thrombosis. Diagnoses were confirmed by objective methods as follows: 1) Peripheral arterial occlusive disease was confirmed by Doppler ultrasonography and angiography; 2) cerebrovascular disease was confirmed by angiography and computed tomography or nuclear magnetic resonance imaging; 3) arterial or venous retinal occlusion was confirmed by retinal fluorescein angiography; 4) deep venous thrombosis was confirmed by ultrasonography or phlebography; 5) caval or pelvic venous thrombosis was confirmed by color flow Doppler imaging; 6) pulmonary embolism was confirmed by scintigraphy or angiography; and 7) myocardial infarction was confirmed by electrocardiography. The control group consisted of 60 apparently healthy persons. These controls were either subjects, students, or members of the hospital staff (30 women and 30 men; mean age SD, 35.8 11.8 years) who were not taking any medications and who were recruited during the study period. Patients who were receiving antiplatelet drugs were not excluded from the investigation. All patients had blood drawn at least 3 months after the occlusive event if they were not receiving heparin or oral anticoagulant treatment. If patients were receiving anticoagulant treatment, laboratory evaluation was done 6 months after the event and at least 1 week after interruption of oral anticoagulant treatment. Therapies known to induce variations in homocysteine levels were not given to any patient before testing. All study participants gave informed consent. Laboratory Studies Venous blood samples were obtained in vacutainer tubes (Becton Dickinson, Rutherford, New Jersey) containing 0.129 M sodium citrate (9:1, v/v) from an antecubital vein and centrifuged within 30 minutes at 2000 g for 10 minutes at room temperature. Plasma aliquots were snap-frozen with methanol and dry ice and stored at 70 C. Assays were done within 3 months of blood collection. Total plasma homocysteine, which corresponds to the sum of free homocysteine, cysteine-homocysteine mixed disulfide, and protein-bound forms, was obtained after cleavage and reduction reactions with sodium borohydride followed by iodoacetic acid treatment. Homocysteine and O-phthaldialdehyde were purchased from Fluka (Buchs, Switzerland). All chemicals and solvents were obtained from BDH (Poole, United Kingdom). High-performance liquid chromatographic analysis was done using the O-phthaldialdehyde precolumn derivatization method [11]. Plasma vitamin B12 and folate levels were determined by using chemiluminometric immunoassays (Ciba Corning ACS, Medfield, Massachusetts). Because heterozygosity for cystathionine -synthase deficiency in patients who have normal fasting plasma homocysteine levels may be detected by an abnormal increment in the total plasma homocysteine level after administration of an oral methionine load [1], all patients older than 14 years were asked to receive oral methionine and have their plasma homocysteine levels measured. Eighty-seven patients (55%) gave their consent. After an overnight fast, a venous blood sample was drawn at 0800 h and L-methionine (0.1 g/kg body weight) was orally administered in 200 mL of fruit juice. Methionine intake was followed by a simple Italian coffee breakfast, and 5 hours later by a regular lunch. In a previous study [12], no diurnal variation in total plasma homocysteine levels in relation to food intake had been observed. The blood specimen was taken 8 hours after methionine intake. In 12 healthy persons, the transient increase in plasma homocysteine level peaked 6 to 8 hours after methionine administration. Increments in plasma homocysteine levels after methionine load were measured in controls. The activity of antithrombin III (Coatest Antithrombin, Chromogenix, Stockholm, Sweden) and plasminogen (Coatest Plasminogen, Chromogenix) were tested by amydolytic methods adapted to the ACL 300 (Instrumentation Laboratories, Milan, Italy). The activity of heparin cofactor II was tested as previously described [13], and protein C and protein S anticoagulant activity [14, 15] and antigens [14, 16] were measured by previously described methods. The anticoagulant response to activated protein C was tested in an activated partial thromboplastin time-based assay using a commercial reagent (Actin FSL, Baxter, Miami, Florida) and human purified protein C activated by the thrombin-thrombomodulin complex [17]. Because this assay was introduced in our laboratory in March 1993, patients who were enrolled in the study before this date were reinvestigated for activated protein C resistance. Family Studies When an abnormality in the laboratory variables was detected, a second blood sample was taken to confirm the observation. In addition, whenever possible, the relatives of the propositus were studied to confirm inheritance of the abnormality. First-degree relatives and siblings of 12 patients with mild hyperhomocysteinemia were studied both while fasting and after methionine intake. For 8 of these families, both parents of the proband were available for study. First-degree relatives and siblings of 11 probands were evaluated for activated protein C resistance and deficiency of protein S, protein C, and plasminogen. Statistical Analysis Results are expressed as mean SD or as median and range. Hyperhomocysteinemia was defined by a persistent elevation of fasting plasma homocysteine levels or by plasma homocysteine levels after methionine load that exceeded the 95th percentile of the distribution in the control group in the presence of normal folate and vitamin B12 plasma levels [5, 18]. In children, hyperhomocysteinemia was diagnosed when fasting plasma homocysteine levels were greater than the 95th percentile of an age-matched control group (12 mol/L [12]). Deficiency of protein C, protein S, and plasminogen and resistance to activated protein C were confirmed by the observation of values persistently lower than the 5th percentile distribution of values in the control group. In the control group, all laboratory variables were normally distributed except total plasma homocysteine levels, which were log-transformed to approximate normal distribution. Homocysteine levels are lower in women than in men [12, 19]. Because sex-related differences in plasma homocysteine levels were also observed in our control group (P < 0.0001), separate 95th percentiles were used for men (fasting levels, 19.5 mol/L; lev

High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).

Variable interference of activated protein C resistance in the measurement of protein S activity by commercial assays

Congenital and acquired deficiencies of vitamin K-dependent protein S (PS) are an established risk factor for thrombotic disease (1,2). Due to the multiple PS domains involved in the interaction with other plasma proteins and the expression of activated protein C (APC) cofactor activity, the existence of qualitative defects of PS is anticipated (3). Thus, measurement of PS anticoagulant activity should be preferred to PS antigens determination in the identification of both congenital and acquired deficiencies. At present, two commercial assays are available for the measurement of the APC cofactor activity of PS. with one of these assays qualitative protein S deficiencies had been erroneously identified (4), as originally indicated by a multicenter collaborative study comparing different functional PS assays (5). In the latter study, plasma samples obtained from patients reportedly affected by qualitative PS deficiencies showed virtual coincidence of free PS antigen and anticoagulant activity levels only when this was evaluated after immunoadsorption of PS (6). This observation raised the possibility of artifacts related to PS testing in whole plasma (5). Following the description of APC resistance a new marker of congenital thrombophilia characterized by defective clotting time prolongation upon additon of APC to plasma (7) -, the seemingly PS deficient patients have been correctly diagnosed as APC resistant cases, with the conclusion that APC resistance may interfere in PS activity testing by the available commercial methods (4). To explore the extent of such interference, we have conducted a prospective comparison of two commercial functional PS assays and our home-made assay (6) in a series of patients with APC resistance referred to our Institution for the evaluation of a thrombophilic state.

Gene-gene and gene-environment interactions in mild hyperhomocysteinemia.

Mild/moderate hyperhomocysteinemia (HHcy), a highly prevalent condition, is independently associated with an increased risk of arterial and venous thromboembolic diseases. Early reports of the association of mild/moderate HHcy with juvenile venous thromboembolism have shown familiarity for HHcy in relatives of index cases with thrombosis. Similar to inherited thrombophilia defects, inheritance of the HHcy phenotype was accordingly retained important for the definition of HHcy as an independent risk factor for thrombosis. A number of common polymorphisms in genes coding for methylenetetrahydrofolate reductase(MTHFR), methionine-synthase, methionine-synthase reductase and cysthationine beta-synthase (CBS) have been explored for their association with homocysteine levels, fasting and post-methionine load, and with thrombotic diseases. MTHFR thermolability accounts for a 10-fold increase in the risk of mild/moderate HHcy. With the possible exception of the CBS844ins68 insertion, there is no evidence for an increased risk of HHcy for any of these polymorphisms, isolated or in association with MTHFR thermolability. Environmental factors and MTHFR thermolability are main determinants of the HHcy phenotype.If mild/moderate HHcy is a pathogenetic risk factor for thrombosis, intervention aimed to improve the vitamin status appears of major importance, irrespective of common gene polymorphisms of the homocysteine metabolism.

Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function

Summary.  Objective: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium‐binding module with a poorly defined functional role. Patients: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. Results: Reverse transcription‐polymerase chain reaction analysis showed the presence of the IVSg‐2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from probands plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C‐independent activities (24–38%) when compared with wild‐type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 ± 30.8 nm, rPSwt; Kd 15.2 ± 0.9 nm) as well as to Ca2+‐dependent conformation‐specific monoclonal antibodies for GLA domain was significantly reduced. Conclusions: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.

Reduced in vivo oxidative stress following 5-methyltetrahydrofolate supplementation in patients with early-onset thrombosis and 677TT methylenetetrahydrofolate reductase genotype

The protective role of folate in vascular disease has been related to antioxidant effects. In 45 patients with previous early‐onset (at age <50 years) thrombotic episodes and the 677TT methylenetetrahydrofolate reductase genotype, we evaluated the effects of a 28d‐course (15 mg/d) of 5‐methyltetrahydrofolate (MTHF) on homocysteine metabolism and on in vivo generation of 8‐iso‐prostaglandin F2α (8‐iso‐PGF2α), a reliable marker of oxidative stress. At baseline, patients’ fasting total homocysteine (tHcy) was 11·5 μmol/l (geometric mean) and urinary excretion of 8‐iso‐PGF2α was 304 pg/mg creatinine, with the highest metabolite levels in the lowest quartile of plasma folate distribution (P < 0·05). After 5‐MTHF supplementation, plasma folate levels increased approximately 13‐fold (P < 0·0001 versus baseline); tHcy levels (6·7 μmol/l, P < 0·0001) and urinary 8‐iso‐PGF2α (254 pg/mg creatinine, P < 0·001) were both significantly lowered, their reduction being proportional to baseline values (r = 0·98 and r = 0·77, respectively) and maximal in patients with the lowest pre‐supplementation folate levels (P < 0·05). The effects on folate (P < 0·0001) and tHcy (P = 0·0004) persisted for at least up to 2 months after withdrawing 5‐MTHF. In parallel with long‐lasting tHcy‐lowering effects, a short‐course 5‐MTHF supplementation reduces in vivo formation of 8‐iso‐PGF2α in this population, supporting the antioxidant protective effects of folate in vascular disease.

Protein S and protein C anticoagulant activity in acute and chronic cardiac ischemic syndromes. Relationship to inflammation, complement activation and in vivo thrombin activity

Protein S (PS) and protein C (PC) anticoagulant activities and thrombin-antithrombin complex (TAT) were measured in 20 patients with AIS, 25 patients with chronic stable angina (CSA) and a control group (C). Although plasma levels of TAT were significantly elevated in patients with CSA (p < 0.01 vs C), they were much higher in patients with AIS (p < 0.001 vs CSA). PC anticoagulant activity was similar in patients and controls. At variance, PS anticoagulant activity was lower in patients with AIS than in those with CSA and controls (p < 0.05), reflecting differences in total PS and C4B-binding protein (C4B-BP) antigen possibly resulting from involvement in the mechanisms of inflammation, complement activation and acute-phase response. The ratios of anticoagulant PS and PC to procoagulant vitamin K-dependent factors IX and II were reduced in AIS patients (0.05 > p > 0.005 vs C). In addition, the ratios of anticoagulant PC and PS to factor IX were lower in patients with AIS than in those with CSA (p < 0.05). These results indicate that in patients with acute ischemic cardiac syndromes the markedly increased in vivo thrombin generation is associated with an unbalance between coagulant and anticoagulant vitamin K-dependent factors.

Erratum: Reduced in vivo oxidative stress following 5- methyltetrahydrofolate supplementation in patients with early-onset thrombosis and 677TT methylenetetrahydrofolate reductase genotype (British Journal of Haematology (2005) 131 (100-106))

The authors of this Full Paper sincerely apologize for the misuse of “standard hydrogen electrode” (SHE) instead of “reversible hydrogen electrode” (RHE) and wish to clarify that all the potentials reported in this paper and the Supporting Information should be against RHE. We also would like to clarify that the electrochemical data was collected versus a Hg/HgO/1.0 m KOH reference electrode during the experiments and had been converted to values vs. RHE, as should be reported. The standard electrode potential of Hg/HgO/1.0 m KOH was 0.098 V vs. SHE based on the manufacturer’s specification. Therefore, in our system, potentials versus RHE were calculated as: