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Dipartimento di Scienze Cliniche e Biologiche, Medicina Interna I (M.T., B.A., F.D., M.V., G.R., A.A.) and Medicina Interna II (S.B., E.B., C.C.), Università di Torino, 10100 Torino, Italy; and Laboratorio Analisi (E.A., G.S.), Azieuda Sanitaria Ospedaliera San Luigi, 10043 Orbassano, Italy
Address all correspondence and requests for reprints to: M. Terzolo, M.D., Medicina Interna I, Azieuda Sanitaria Ospedaliera San Luigi, Regione Gonzole, 10, 10043 Orbassano, Italy. E-mail: terzolo{at}usa.net.
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Abstract |
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Introduction |
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It has been suggested that elevated homocysteine concentrations may be an independent risk factor for cardiovascular disease and venous thrombosis. A common polymorphism exists for the gene that encodes the methylene tetrahydrofolate reductase (MTHFR), an enzyme required for the folate-dependent remethylation of homocysteine to methionine. The remethylation pathway regulates fasting levels of homocysteine, and individuals who have a cytosine-to-thymidine substitution at base 677 of the gene encoding for MTHFR, which results in an amino acid change alanine 222 valine, have also reduced enzyme activity, and higher homocysteine (7, 8) and lower folate levels than those without this substitution (9).
The initial epidemiological evidence in support of the hypothesis that elevated concentrations of homocysteine are associated with cardiovascular disease came from retrospective case-control studies (10, 11, 12), whereas inconsistent results were reported from prospective observational studies, in which blood for homocysteine measurements were collected before the clinical onset of disease (13). However, a recent metaanalysis of 30 studies involving 5000 ischemic heart disease events and 1000 stroke events, which focused particularly on the results from prospective studies, confirmed that total blood homocysteine concentrations are associated with the risk of ischemic heart disease and stroke (14). An accompanying metaanalysis of 40 studies of MTHFR polymorphism demonstrated that individuals with the TT polymorphism (individuals who are homozygotes for the C-to-T substitution) have a higher risk of ischemic heart disease than those with the CC polymorphism (15).
Moreover, another recent metaanalysis of MTHFR studies showed a significantly higher risk of both ischemic heart disease and deep vein thrombosis (with or without pulmonary embolism) in people with the C-to-T mutation. In the same study, a metaanalysis of prospective studies was also performed, which showed a significant association between homocysteine concentration and ischemic heart disease similar in size to that expected from the results of the MTHFR studies and a significant association with stroke (16). The concordance between the risk estimates obtained in these metaanalyses provides compelling evidence for a causal association between mild hyperhomocysteinemia and either cardiovascular disease, involving coronary as well as peripheral arteries, or deep vein thrombosis in the general population.
Resistance to activated protein C caused by a single point mutation in the factor V gene (factor V Leiden) (17) and the G-to-A substitution in the 3' untranslated region of the prothrombin gene (G20210A), which is associated with an increased factor II activity (18), are the most common hereditary risk factors for venous thrombosis.
Therefore, we thought it of interest to evaluate circulating homocysteine concentrations in patients with Cushing’s syndrome along with DNA analysis for detection of C677T polymorphism in the MTHFR gene as well as other thrombophilic genotypes (factor V Leiden and the G20210A variant of the prothrombin gene).
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Subjects and Methods |
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Subjects were drawn from a series of patients with overt Cushing’s syndrome referred to our center from 1996 to 2002. There were 41 consecutive patients (14 men and 27 women) aged 48.2 ± 18.4 yr. Twenty-five patients had pituitary-dependent Cushing’s syndrome, or Cushing’s disease, six patients had Cushing’s syndrome caused by ectopic ACTH secretion, and 10 patients had adrenal-dependent Cushing’s syndrome sustained by cortisol-secreting adrenocortical adenoma (n = 5) or ACTH-independent macronodular adrenal hyperplasia (n = 5). The ectopic ACTH secretion was sustained by well-differentiated endocrine tumors without cellular atypia or signs of invasion of contiguous structures, or metastases, and showing two or less mitoses per 10 high-power fields and 2% or less Ki-67 positive cells. The patients with Cushing’s syndrome caused by adrenal carcinoma (n = 2) or ACTH-secreting malignancy (n = 2) were excluded because elevated homocysteine concentrations have been reported in association with several types of malignant tumors (19). Moreover, three patients with Cushing’s disease were excluded because they reported current or recent use of drugs interfering with homocysteine levels (i.e. anticonvulsants, multivitamin preparations) (20). The 41 patients were evaluated in an active phase of the disease, and none of them was receiving any drug known to affect the hypothalamic-pituitary-adrenal axis. Sixteen patients (three men and 13 women aged 45.4 ± 13.9 yr) who were in remission for at least 1 yr after successful surgery were also evaluated; they had discontinued replacement therapy for at least 6 months. In this group of patients, the etiology of Cushing’s syndrome was pituitary dependent in 12 subjects, adrenal-dependent in two subjects, and ectopic ACTH secretion in two subjects. Only 10 patients with cured Cushing’s syndrome were evaluated also in an active phase of the disease.
The diagnosis of Cushing’s syndrome was made on the basis of clinical features and standard hormonal and radiologic criteria (21). The inferior petrosal sinus sampling in conjunction with CRH stimulation was performed in 12 patients who had negative pituitary magnetic resonance imaging or magnetic resonance imaging evidence of a pituitary mass along with discordant results of the noninvasive tests. Published criteria for central to peripheral ACTH gradients have been used to diagnose a nonpituitary source of ACTH (22). For Cushing’s disease, the diagnosis was confirmed by finding a pituitary adenoma with positive ACTH staining at pathological examination or by the occurrence of postoperative adrenal insufficiency that lasted for at least 6 months. In all cases of ectopic ACTH syndrome and adrenal-dependent Cushing’s syndrome, the diagnosis was histologically confirmed. A group of 105 subjects (39 men and 66 women aged 44.3 ± 14.2 yr) recruited from blood donors, who were matched for gender distribution and age, served as controls. All these subjects were not taking any medication or multivitamin supplements; their physical exam and standard biochemical and radiological evaluation excluded relevant diseases.
The study was designed in agreement with the Declaration of Helsinki and was approved by the local Ethical Committee. The patients and subjects volunteered for the study and gave their informed consent.
Methods
The patients and controls underwent physical examination; chest radiograph; electrocardiogram; routine laboratory evaluation; and measurement of serum homocysteine, serum folate, serum vitamin B12, plasma antithrombin, plasma protein C, plasma protein S concentrations, and activated protein C (APC) resistance. All patients underwent an endocrine work-up aimed to assess the function of the hypothalamic-pituitary-adrenal axis, as previously reported (23). Briefly, the patients underwent measurement of 1) serum cortisol at 0800 and 1200 h, 2) 24-h excretion of urinary free cortisol, 3) serum cortisol after overnight 1 and 8 mg dexamethasone, and 4) serum cortisol and plasma ACTH after ovine CRH test. Premenopausal women were studied in the early follicular phase of the menstrual cycle.
Assays
Blood samples were drawn at 0800 h, after overnight fast, from an antecubital vein that was cannulated 30 min before. Serum homocysteine levels were measured by HPLC using a reversed-phase analytical cartridge and fluorescence detection (Bio-Rad Laboratories, Munich, Germany). The fluorescence signal was detected by means of a HP1046A fluorometer (Hewlett Packard, Portland, OR) operating at an excitation wavelength of 385 nm and emission wavelength of 515 nm. Quantitation accuracy was granted by the use of an internal standard (acetyl-cysteine) and the intra- and interassay coefficients of variation were between 3.9–4.6 and 5.6–6.4%, respectively, in the concentration range between 8.7 and 26.9 µmol/liter. The sensitivity of the assay was 0.5 µmol/liter. Serum folate and vitamin B12 levels were determined using an automated chemiluminescence system (Bio-Rad Laboratories). Determination of antithrombin, protein C and protein S concentrations, and APC resistance was made using commercially available reagents (Bio-Rad Laboratories).
Hormonal variables were measured by RIA or immunoradiometric assay methods, using commercially available kits, as previously described (23). All samples for an individual subject were determined in the same laboratory, in a single assay, and in duplicate. Intra- and interassay coefficients of variation for all hormone variables were less than 8 and 12%, respectively. Routine clinical chemistry variables were determined using standard enzymatic methods.
Molecular studies
Genomic DNA was prepared from peripheral blood cells using standard methods. PCR was performed on a Thermal Cycler (New England Biolabs, Beverly, MA) using 12.5-pmol primers and 0.5 U Taq polymerase in a final volume of 50 ml. PCR conditions were: denaturation at 94 C for 30 sec, annealing from 55 to 69 C for 30 sec, and extension at 72 C for 30 sec. Oligonucleotide primers for the different genes were as described in the corresponding references or obtained by public databases (http://www.ncbi.nlm.nih.gov/).
To assess factor V R506G and factor II G20210A mutations, two fragments were amplified corresponding to exon 10 of factor V and 3'untranslated region of factor II genes; the amplified products were digested with MnII and HindIII endonucleases (New England Biolabs), respectively, according to the manufacturer’s recommendations. The restriction fragments were separated by electrophoresis on agarose gel and visualized by ethidium bromide staining.
A similar procedure was used for detection of C677T polymorphism in the MTHFR gene using for restriction analysis HinfI and BsrI (New England Biolabs) restriction enzymes, respectively. The three genotypes of MTHFR were defined as follows: CC, normal homozygous; CT, heterozygous; and TT, mutant homozygous.
The factor V R506G and factor II G20210A mutations were simultaneously detected by the CVD strip assay kit (Nuclear Laser Medicine, Settala, Milan, Italy) in 32 patients and 41 controls.
Statistical analysis
Database management and all statistical analyses were performed by using the Statistica for Windows software package (Statsoft Inc., Tulsa, OK). Rates and proportions were calculated for categorical data and means and SDs for continuous data; 95% confidence intervals were always provided. Normality of data was assessed by the Wilk-Shapiro’s test. For continuous variables, differences were analyzed by means of the two-tailed Student’s t test when data were normally distributed and using the Mann-Whitney U test for nonparametric data. Analysis of covariance with Tukey’s post hoc comparison of the means was used for multiple comparisons. For categorical variables, differences were analyzed by means of the 
2 test and Fisher’s exact test when appropriate. Correlation analyses were determined by calculating the Spearman’s R coefficient. Bonferroni adjustment for multiple comparisons was performed as appropriate. In addition, multiple regression analysis was performed to determine which variables predicted serum homocysteine concentrations in patients with Cushing’s syndrome. The candidate predictive variables were selected from a matrix of simple correlations between homocysteine and factors known to influence its concentrations (serum folate, serum vitamin B12, serum creatinine, fasting glucose, systolic and diastolic blood pressure); midnight cortisol was also selected as the most sensitive marker of endogenous hypercortisolism (24). For the distribution of MTHFR genotypes, Hardy-Weinberg equilibrium was assessed by 2 analysis. Levels of statistical significance were set at P < 0.05.
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Results |
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and 2. None of the patients had a deficiency of the natural anticoagulants antithrombin, protein C, and protein S, and only one patient had APC resistance. A statistically significant difference in homocysteine levels was found among the three groups (P < 0.0001). The patients with active Cushing’s syndrome had significantly higher serum homocysteine levels than either the patients in remission (P = 0.006) or controls (P = 0.0001), whereas serum homocysteine levels were not statistically different between the patients in remission and controls (Fig. 1 and Table 2). Serum folate concentrations were lower in the patients with active Cushing’s syndrome than either the patients in remission or controls (Fig. 2 and Table 2). The difference between the group of patients with active Cushing and that of controls was statistically significant (P < 0.0001). Serum vitamin B12 concentrations did not differ among the three groups (Table 2).
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In control subjects, homocysteine levels were significantly higher in men than in women [13.4 ± 4.0 (12.1–14.7) vs. 11.7 ± 4.3 µmol/liter (10.6–12.8), P = 0.02], whereas a specular pattern was observed for serum folate levels [6.2 ± 2.7 (5.4–7.1) vs. 7.9 ± 2.6 ng/ml (7.2–8.5), P = 0.002] [14.0 ± 6.1 (12.2–16.1) vs. 17.9 ± 5.9 nmol/liter (16.3–19.2), P = 0.002]; no gender-related difference was observed in patients with active Cushing’s syndrome for either homocysteine [18.0 ± 3.0 (16.3–19.8) vs. 17.7 ± 4.6 µmol/liter (15.8–19.5), P = NS] or folate concentrations [4.1 ± 1.8 (3.0–5.1) vs. 5.5 ± 3.3 ng/ml (4.0–6.9), P = NS] [9.3 ± 4.1 (6.8–11.5) vs. 12.5 ± 7.5 nmol/liter (9.1–15.6), P = NS]. The separate comparison of women and men reproduced the same differences between hypercortisolemic patients and controls.
In patients with Cushing’s syndrome, the best model generated by multiple regression analysis accounted for 28% (r2) of the total variation of serum homocysteine (P = 0.002). In this model, predictors of homocysteine concentrations were midnight cortisol (beta = 0.33, P = 0.01) and serum folate (beta = –0.32, P = 0.02).
The distribution of MTHFR genotypes in the whole population was compatible with the Hardy-Weinberg equilibrium. Table 3 indicates the frequency distribution of the genotypes between patients and controls; no significant difference was observed. In control subjects, the TT homozygotes had significantly higher serum homocysteine concentrations as compared with the CC or CT genotypes. However, the latter genotype had values that were not statistically different from the CC genotype (Fig. 3). Serum folate levels showed an inverse relationship with the MTHFR genotypes. In the patients with active Cushing’s syndrome, higher homocysteine concentrations were observed in the TT homozygotes, compared with the other genotypes, whereas no difference in serum folates was observed (data not shown). No difference in homocysteine and folate concentrations was detected among different MTHFR polymorphisms in the patients with inactive Cushing’s syndrome (Fig. 3). To allow for the independent contribution of MTHFR genotype-to-homocysteine concentrations, a separate comparison between patients and controls with any given genotype was also performed. A statistically significant between-group difference was confirmed for any polymorphism (P = 0.001). Regardless of the MTHFR genotype, the patients with active Cushing’s syndrome had higher serum homocysteine concentrations than either other group (Fig. 3).
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Discussion |
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At present, no data are available in literature on serum homocysteine levels and MTHFR polymorphism in patients with Cushing’s syndrome. Mild hyperhomocysteinemia is an established risk factor for atherosclerosis and vascular disease (20). Moderate increases in serum homocysteine occur as a result of a mutation in the gene encoding for the enzyme MTHFR in which cytosine is replaced by thymidine (C-to-T) at base position 677 of the gene. This variant of the enzyme has reduced activity (27), resulting in an elevation of serum homocysteine concentrations of about 20% (8). The C-to-T mutation is surprisingly common, with about 16–21% of people in the Italian population being homozygous TT, 23–32% homozygous CC, and 47–60% heterozygous CT (28, 29, 30, 31).
Interindividual variation in homocysteine levels is an example of genetic-environmental interaction, in which dietary habits play an important role (32) because the TT genotype has little effect on homocysteine levels when folate consumption is high (27). A number of other factors influence homocysteine metabolism, including several disease states and medications. Homocysteine levels increase with elevations in creatinine and are typically elevated in chronic renal failure (33). A modest association among hyperinsulinemia, reflecting insulin resistance, and fasting levels of homocysteine has been found in the population-based Framingham Offspring Study. In that study, homocysteine levels were higher among subjects with specific features of the insulin resistance syndrome, especially hypertension and obesity (34). Among the other factors associated with hyperhomocysteinemia, there are hypothyroidism (35) and cigarette smoking (36).
The results of the present study demonstrate for the first time that Cushing’s syndrome is associated with hyperhomocysteinemia, whereas the patients in remission have homocysteine concentrations comparable with healthy subjects. Hypercortisolemic patients also showed reduced folate concentrations in comparison with control subjects, a finding that does not appear explainable with peculiar dietary habits. To the best of our knowledge, it has never been reported that sustained cortisol excess is able to interfere with folate metabolism.
In the present study, several lines of evidence support the view that hypercortisolism is an independent factor contributing to hyperhomocysteinemia in Cushing’s syndrome. First, reduced folates concentrations do not fully account for hyperhomocysteinemia in active Cushing’s syndrome, which was confirmed after statistical adjustment for the difference in folates. Patients with active Cushing had higher homocysteine concentrations than either other group even when the comparison was limited to subjects with serum folate above the mean levels observed in our study sample. Second, the frequency of the TT genotype among patients was not higher than that observed in the control group and was overall comparable with that reported in the Italian population (28, 29, 30, 31). Moreover, the patients with Cushing’s syndrome in remission had lower homocysteine concentrations than patients with active Cushing’s syndrome. Third, we performed separate comparisons of the three groups of subjects (active Cushing, inactive Cushing, and controls) for any MTHFR genotype, and we found that the patients with active Cushing’s syndrome had higher homocysteine concentrations than either other group, regardless of the MTHFR genotype. Fourth, midnight serum cortisol was found to be a predictor of homocysteine concentrations along with serum folate concentrations in a multiple regression model. The relationship with midnight serum cortisol is very interesting because it is admittedly considered the most sensitive marker of endogenous glucocorticoid excess (24, 37).
We disclose the limits of the case-control design of our study that may be susceptible to confounding. However, we do not think of having inadvertently selected a control group with homocysteine concentrations lower than the general population because the mean homocysteine level (12.3 µmol/liter) in our sample of control subjects was comparable with values previously observed in the Italian population (28, 29, 30, 31). Even if statistical adjustment was performed to control for differences in the relevant parameters between patients and controls, we cannot exclude that hyperhomocysteinemia of Cushing’s syndrome is partially explained by some metabolic and vascular consequences of sustained cortisol excess. However, this does not reduce the clinical importance of our results.
Hyperhomocysteinemia may play a role in the pathogenesis of the prothrombotic state of Cushing’s syndrome and may contribute to the increased cardiovascular risk of these patients (1, 2, 3, 4). Interestingly, our patients with inactive Cushing’s syndrome did not show a complete normalization of their adverse cardiovascular risk profile, notwithstanding hypercortisolism was resolved. This finding is not surprising because it was already reported that an increased cardiovascular risk persists after 5 yr of successful cure in patients with Cushing’s disease (38). In our study, patients with Cushing’s syndrome in remission showed significantly reduced homocysteine concentrations than patients with active disease, even if body mass index, blood pressure values, and fasting glucose and lipid levels did not differ significantly between the two groups. This finding strengthens the relationship between hypercortisolism and hyperhomocysteinemia.
The issue of reverse causality, i.e. the possibility that elevated homocysteine concentrations occur as the result of preexisting vascular disease (14), does not seem relevant to our data because previous arterial thrombotic events were recorded in a minority of our patients, and the elevation in homocysteine concentrations in the patients with Cushing’s syndrome was confirmed after excluding such subjects.
The factor V Leiden mutation (17) and the G20210A mutation of factor II, prothrombin (19), which are well-established genetic risk factors for thrombosis, were observed in our sample of patients with the same frequency than in the general population. These data make unlikely the possibility that hypercoagulability of Cushing’s syndrome is due to a linkage with these genetic traits. However, these inherited determinants may interact with hypercortisolism to increase the risk of thrombosis, as was the case in a patient of our sample.
In conclusion, our data suggest that endogenous hypercortisolism is associated with hyperhomocysteinemia and reduced folate concentrations, whereas the patients in remission have homocysteine concentrations comparable with healthy subjects. Low folate concentrations do not fully account for the increase in homocysteine levels that are positively correlated with cortisol levels. Hyperhomocysteinemia may be key to the prothrombotic state and increased cardiovascular risk of patients with Cushing’s syndrome. The available evidence suggests that the degree of elevation of homocysteine levels demonstrated in the patients with Cushing’s syndrome may be predictive of a significant risk of cardiovascular or cerebrovascular events and venous thrombosis as well. In the general population, the odds ratio for a 5 µmol/liter increase in serum homocysteine concentrations, which is comparable with the difference observed between our patients and controls, were, for ischemic heart disease, 1.32 (1.19–1.45); for stroke, 1.59 (1.29–1.96); and, for deep vein thrombosis, 1.60 (1.15–2.22), as reported in a metaanalysis of genetic and prospective studies (16). Moreover, two recent metaanalyses (14, 16) demonstrated that lowering homocysteine concentrations by 3 µmol/liter from current levels (achievable by increasing folic acid intake) would reduce the risk of ischemic heart disease by 11–16%, deep vein thrombosis by 25%, and stroke by 19–24%. Prospective studies of the effects on vascular disease of lowering homocysteine with folic acid supplementation should provide further information about the relevance of homocysteine levels to the risks of arterial and venous thrombosis in Cushing’s syndrome.
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Acknowledgments |
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Footnotes |
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Abbreviations: APC, Activated protein C; MTHFR, methylene tetrahydrofolate reductase.
Received January 16, 2004.
Accepted May 10, 2004.
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T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA 288:2023–2031This article has been cited by other articles:
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  B. Van Zaane, E. Nur, A. Squizzato, O. M. Dekkers, M. B. Twickler, E. Fliers, V. E. A. Gerdes, H. R. Buller, and D. P. M. Brandjes Hypercoagulable State in Cushing's Syndrome: A Systematic Review J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 2743 - 2750. [Abstract] [Full Text] [PDF]
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P. Anagnostis, V. G. Athyros, K. Tziomalos, A. Karagiannis, and D. P. Mikhailidis The Pathogenetic Role of Cortisol in the Metabolic Syndrome: A Hypothesis J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 2692 - 2701. [Abstract] [Full Text] [PDF]
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 P Borrione, M Rizzo, A Spaccamiglio, R A Salvo, A Dovio, A Termine, A Parisi, F Fagnani, A Angeli, and F Pigozzi Sport-related hyperhomocysteinaemia: a putative marker of muscular demand to be noted for cardiovascular risk Br. J. Sports Med., November 1, 2008; 42(11): 894 - 900. [Abstract] [Full Text] [PDF]
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A. Faggiano, D. Melis, R. Alfieri, M. De Martino, M. Filippella, F. Milone, G. Lombardi, A. Colao, and R. Pivonello Sulfur Amino Acids in Cushing's Disease: Insight in Homocysteine and Taurine Levels in Patients with Active and Cured Disease J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6616 - 6622. [Abstract] [Full Text] [PDF]
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D. J. Brotman, J. P. Girod, M. J. Garcia, J. V. Patel, M. Gupta, A. Posch, S. Saunders, G. Y. H. Lip, S. Worley, and S. Reddy Effects of Short-Term Glucocorticoids on Cardiovascular Biomarkers J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3202 - 3208. [Abstract] [Full Text] [PDF]
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