- Email: [email protected]
Prevalence of hyperhomocysteinemia in an elderly population
AJH
2002; 15:394 –397
Prevalence of Hyperhomocysteinemia in an Elderly Population Jorge J. Janson, Carlos R. Galarza, Alicia Muru´ a, Irene Quintana, Pablo A. Przygoda, Gabriel Waisman, Luis Camera, Lucia Kordich, Margarita Morales, Luis M. Mayorga, and Mario I. Camera Background: Currently, total hyperhomocysteinemia (tHHcy) is a well-known condition linked to a higher risk of vascular disease. Prevalence of HHcy increases in elderly persons as the risk associated with it persists. Because factors can be potentially reduced in the elderly, it is important to carry out epidemiologic studies of HHcy. Procedure: Previously we described the prevalence of hypertension control in an elder population; now, in an observational cross-sectional simple blind study, total homocysteine (tHcy) concentration was determined in 196 of 400 patients from the original cohort. Results: Mean Hcy concentration was 13.2 mol/L (95% confidence interval 12.4 –14.0; range, 5.0 to 48.9); 15.0 mol/L for men and 12.3 mol/L for women. Mean serum folic acid levels were 4.9 ⫾ 3.1 ng/mL (range, 2.0
S
everal epidemiologic prospective and case-control studies supported the observation that high levels of homocysteine (Hcy) are an important risk factor in arteriosclerotic vascular disease affecting coronary, cerebral, and peripheral arteries.1,2 Risk increases linearly, and each basal tHcy increase of 5 mol/L in fasting subjects is associated to a relative risk of vascular arteriosclerotic disease of 1.35 (95% confidence interval [CI] 1.1–1.6) for men and 1.42 (95% CI 0.99 –2.55) for women.3 Homocysteine levels increase significantly with age, and total hyperhomocysteinemia (tHHcy) prevalence has been reported to be higher in the elderly than in other age groups.4 – 6 However, it is still unknown whether it is due to nutritional causes or is related to metabolic changes common to old age and poor nutritional absorption. The purpose of this study was to evaluate the levels of tHcy in elderly persons living in the city of Buenos Aires, Argentina. Received February 12, 2001. First Decision December 27, 2001. Accepted February 13, 2002. From the Sociedad Argentina de Medicina Vascular (JJJ, CRG, AM, MM, MIC); Facultad de Ciencias Exactas y Naturales, UBA (JJJ, CRG, IQ, LK); and Hospital Italiano de Buenos Aires, Servicio de Clinica 0895-7061/02/$22.00 PII S0895-7061(01)02165-3
to 20.0 ng/mL), and vitamin B12 levels were 384.8 ⫾ 314.1 pg/mL (range, 48.0 to 1500.0 pg/mL). Taking into account the reference values established by the Third National Health and Nutrition Examination Survey III study, HHcy was detected in 69.8% of all the subjects evaluated. The study showed that 76.2% of the men and 66.4% of the women had high Hcy levels. Conclusions: The very high prevalence of tHHcy in the elderly population, and the consequent risks associated with it suggest that although there are no trials that effectively prove the benefit of tHcy decrease, nutritional intervention is still justified. Am J Hypertens 2002;15: 394 –397 © 2002 American Journal of Hypertension, Ltd. Key Words: Homocysteine, elderly people, prevalence, epidemiologic studies.
Methods Subjects An observational, simple blind study was carried out in 202 individuals older than 65 years, from an original sample of 400 patients. These subjects were randomly chosen from a group of 35,800 elderly people from the Unit No. 2 of the National Health Care Program called PAMI (Programa de Atencio´n Me´dica Integral) of the Hospital Italiano de Buenos Aires, who had participated in a previous study.7 Eventually samples from 196 subjects were eligible for Hcy determination. All subjects were evaluated on the basis of a survey of symptoms related to coronary, cerebral, and peripheral diseases. Data were completed with a physical examination, an electrocardiogram (ECG), and a questionnaire on risk factors (smoking, alcohol, stress, sedentary lifestyle). Participants were also asked about the use of vitamin supplementation and drugs, as well as about other causes Medica y Unidad de Hipertension arterial, Buenos Aires, Argentina. Address correspondence and reprint requests to Dr. Jorge J. Janson, Unidad de Hipertension Arterial, Hospital Italiano de Buenos Aires, Gascon 450 (1181), Buenos Aires, Argentina; e-mail: [email protected] © 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
AJH–May 2002–VOL. 15, NO. 5
HYPERHOMOCYSTEINEMIA IN AN ELDERLY POPULATION
of tHHcy. In subjects with previous diagnoses of vascular disease, objective data on these diagnoses were included. Patients with the following pathologies were considered vascular disease carriers. Peripheral arteriopathy: Subjects having undergone aortofemoral or femoropopliteal bypass surgery or with previous intermittent claudication and tests showing a decrease in arterial flow in the lower limbs (Doppler echography, arteriography, or plethysmography). Ischemic cerebral disease: Subjects previously having temporary motor or sensitive focal neurologic deficit, or with sequel and computerized axial cerebral tomography without signs of hemorrhage, and subjects with or without symptoms with Doppler echography of carotid arteries with obstructions higher than 20%. Coronary pathologies: Subjects having undergone myocardial revascularization surgery or angioplasty, or subjects with or without previous thoracic pain and ECG sequel necrosis, or with previous thoracic pain with positive complementary tests for ischemia (ergometry, ventriculogram), and subjects with previous thoracic pain and under antiangina medication with no complementary tests. Informed consent to participate in the study was obtained from all subjects according to guidelines set out by our institutional review board. Blood Samples Blood samples from fasting subjects were obtained using EDTA as anticoagulant. Samples were immediately chilled at ⬍4°C and centrifuged (2000 ⫻ g, 10 min) within 1 h of collection. Plasma was immediately frozen at ⫺70°C until Hcy determination. Serum samples were obtained and stored at ⫺70°C until analysis. Laboratory Determinations Total plasma Hcy levels were determined by enzyme immunoassay (Axis Biochemicals ASA, Oslo, Norway). This method was validated by our research group.8 Serum folic acid and vitamin B12 were determined in the sera of 70 patients by a combined system of competitive immunoassay and chemiluminescence (AccessSanofi-Pasteur; Access Beckman Coulter, Chaseka, MN). This procedure involved the use of monoclonal antibodies, paramagnetic particles, and a chemiluminescence substrate. The light emitted, which was inversely proportional to the concentration of folic acid or vitamin B12, was registered. The study of each patient was completed with the determination of total cholesterol, triglycerides, HDL-cholesterol, and creatininemia (applying the usual laboratory techniques). Statistical Methods Homocysteine, folic acid, and vitamin B12 showed a bias in the distribution of frequencies. Therefore, we performed a logarithmic transformation (base 10) to obtain an adequate normal distribution for the application of parametric
395
Table 1. General characteristics of the population % Women Sedentary Diabetes Widow/widower Married Patients with vascular disease Patients with coronary disease
68.4 64.3 17 33 48.9 17.3 10.2
tests. Thus, when we refer to the mean of these variables, we use the geometric mean (⫾95% CI). Results are expressed as mean ⫾ SD if nothing else is specified. Pearson correlation coefficients and probabilities were calculated for the relationship between HHcy and continual quantitative variables (folic acid, vitamin B12, creatininemia, age, etc.). The analyses of differences between the groups with and without vascular disease were performed by Student t test for two-tailed independent samples. Frequency distribution of nominal variables was analyzed for different values of HHcy applying the Fisher’s exact test and the 2 test. A multivariate analysis (logistic regression) was carried out taking vascular disease as the dependent variable, and different known risk factors plus Hcy as the continual and dichotomic variable with two different values (12 to 15 mol/L).
Results In the group studied (62 men and 134 women), mean age for the patients was 74.4 years (range, 66 to 93 years). General characteristics of the population are shown in Tables 1 and 2. Total plasma tHcy concentration was measured in 196 subjects from the group already described. Mean concentration was 13.2 mol/L (95% CI 12.4 –14.0; range, 5.0 to 48.9 mol/L), 15.0 mol/L (95% CI 13.6 –16.7; fifth percentile 7.0 mol/L and 95th percentile 30 mol/L) for men, and 12.3 mol/L (95% CI 11.5–13.2; fifth percentile 7.2 mol/L and 95th percentile 21.5 mol/L) for women. As it was expected, the differences in total Hcy between sexes were statistically significant (P ⬍ .05). Homocysteinemia correlated weakly with age (r ⫽
Table 2. Clinical characteristics of the population Media (SD) 2
BMI (kg/m ) Creatinine (mg/dL) Cholesterol (total) (mg/dL) HDL (mg/dL) Tg (mg/dL)
27.31 1.05 234.14 48.79 125.23
(4.74) (0.18) (39.86) (7.77) (60.6)
BMI ⫽ body mass index; HDL ⫽ high-density lipoprotein; Tg ⫽ triglyceride.
396
HYPERHOMOCYSTEINEMIA IN AN ELDERLY POPULATION
0.195), creatinine (r ⫽ 0.304), body mass index (r ⫽ 0.221), and folic acid (r ⫽ ⫺0.435). In 34 patients, previous obstructive vascular disease was detected (20 coronary disease, 11 peripheral arteriopathy, 10 previous ischemic cerebrovascular disease, and 3 carotid disease). Ten of the 34 patients had symptomatic atheromatose disease in more than one location. From these, 45% had at least another vascular disease risk factor, apart from increased levels of Hcy. Mean tHHcy in vascular patients (14.4 mol/L; 95% CI 12.6 –16.5) was 14.2% higher than in nonvascular subjects (12.7 mol/L; CI 95% 11.8 –13.6). This difference, however, was not significant. Vitamin Levels Vitamins were measured in sera from 70 patients chosen at random. Serum folic acid level was 4.9 ⫾ 3.1 ng/mL (range, 2 to 20 ng/mL) and vitamin B12 level was 384.8 ⫾ 314.1 pg/mL (range, 48 to 1500 pg/mL). Low levels of vitamin B12 (⬍180 pg/mL) were observed in 14.7% of the subjects, and low levels of plasma folic acid (⬍3 ng/mL), in 24.7% of the subjects, independently of tHHcy. Among individuals with high tHcy levels, deficits of vitamin B12 and folic acid were 15.38% and 32.7%, respectively. Conversely, 94% (n ⫽ 17) of the subjects with low folic acid, and 90% (n ⫽ 9) of the subjects with vitamin B12 deficit showed high tHcy levels.
Discussion Because the association between tHHcy and risk of vascular events is gradual and continual, and tHcy levels are highly prevalent,9 it is still difficult to establish reference values. Recently, the Third National Health and Nutrition Examination Survey (NHANES III) study10 established that higher values (95th percentile) in a healthy, young population (aged 20 to 39 years) with an adequate vitamin plasma level and without renal insufficiency can be considered having tHHcy. On the basis of these criteria, tHHcy was defined for values higher than 11.4 mol/L in men and 10.4 mol/L in women. Taking into account the values considered normal for the American group evaluated by the NHANES III study, our study showed a very high prevalence of tHHcy in a randomly chosen group of elderly people living in the city of Buenos Aires. Thus, 69.8% of the subjects were considered to have tHHcy. The proportion of tHHcy in men (76.2%) was significantly higher than in women (66.4%). On the other hand, tHHcy prevalence in American individuals older than 60 years old was 43.2% and 46.5% for men and women, respectively. Several studies reported that plasma tHcy levels increase with age, depending on nutritional and metabolic factors, and on changes in vitamin B12 absorption and renal excretion. In our country, eating habits are characterized by high consumption of animal proteins and low ingestion of food
AJH–May 2002–VOL. 15, NO. 5
rich in folates. In addition, there is a high prevalence of the thermolabile variant of the methylene tetrahydrofolate reductase in our population. In a cohort of elderly survivors from the original population of Framingham, mean tHcy concentration was 11.9 mol/L, values higher than 14 mol/L (90th percentile in this population) were detected in 29.3% of the subjects.11 On the basis of these criteria, our study reported 44.5% of elderly patients with high Hcy levels. On the other hand, values ⱖ9 mol/L, which was the lowest mortality level in the group studied by Nygard et al,12 were found in 79% of our cases. Subjects defined as hyperhomocysteinemic (NHANES III criteria) in our study also showed high prevalence of vitamin deficit, 15.38% for vitamin B12 and 32.7% for folic acid. Although the relative importance of vitamin deficits to determine Hcy level decreases with age (population attributable risk), it has been reported that between one third and two thirds of elderly people with high tHcy levels also have vitamin deficits. Folic acid deficit is considered the essential cause of high tHcy concentration in young people, whereas the B12 deficit increases with aging.13 Consequently, the treatment of tHHcy in elderly people requires an increase in the consumption of food with high content of folates and vitamin B12. Moreover, if pharmacologic supplementation is added, it would be advisable to measure vitamin B12 or administer orally high doses of B12 together with folic acid (approximately 1% is absorbed in cases of atrophic gastritis). Assuming that no selection bias was present in our study, the probability of obtaining an “ideal” median, 9 mol/L, is less than one in a million (z ⫽ ⫺12.81). As in the case of other cardiovascular risk factors such as cholesterol or arterial hypertension, the relative risk added by tHcy decreases with age. In the past, this led to the underestimation of the impact of risk factors in elderly people, with the consequent disregard for their treatment. This situation has been reversed, not only understanding the significant differences between relative and absolute risks, but also gathering evidence to demonstrate that the association between the factor (ie, tHcy) and the vascular event continues with age. In the Rotterdam study,14 analyses carried out on a cohort of 7983 residents, all older than 55 years at the beginning of treatment, showed increases between 5% and 10% for the occurrence of different vascular events per each 1 mol/L tHcy increase. In a 4-year follow-up, participants of the upper quintile with levels higher than 18 mol/L showed a significant increase of myocardial infarction risk (odds ratio, 2.4; CI 95% 1.1–5.4) and cerebrovascular accident (odds ratio 2.5; CI 95% 1.2–5.4) when compared with those from the lower quintile with values less than 12.0 mol/L. In a recent analysis on 10-year mortality in elderly people in Framingham (n ⫽ 1933), values of tHcy higher than 14.3 mol/L were associated to a nonadjusted rela-
AJH–May 2002–VOL. 15, NO. 5
HYPERHOMOCYSTEINEMIA IN AN ELDERLY POPULATION
tive risk of cardiovascular death of 2.2 (95% CI 1.7–2.8) in respect of lower values.10,15 The European Concert Project studied the interaction between tHcy and other cardiovascular risk factors, finding important implications for risk management. This study deserves special consideration for the interaction between tHcy and hypertension, and tHcy and smoking, because they are more than multiplicative. The study reported an odds ratio of 11 for the interaction between fasting tHcy and hypertension and an odds ratio of 4.6 for fasting tHcy and smoking.16 The subjects from our study showed, in addition to the high tHHcy prevalence (69.8%), a high proportion of cases with arterial hypertension (77.5%). Eight percent of our subjects were smokers. This is a group with high global vascular risk. From other point of view, tHcy might be a causal factor for elevated blood pressure.17 Therefore, according to previous data, just improving eating habits in elderly people could bring significant benefits.18 However, it is important that simultaneous prospective intervention studies were carried out to evaluate the impact of tHcy decrease in the morbidity and mortality due to atherothrombotic disease. Reports on these data are particularly important in the case of elderly people with high absolute risk of vascular disease, in whom intervention studies on other risk factors proved successful.19
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgments The following investigators participated and are gratefully acknowledged: Martin O’Flaherty, Carolina Ponce de Leon, Leonardo Garfi, Oscar Lopez, and Alejandro Manzur.
References 1. 2.
3.
4.
5. 6.
Kullo IJ, Gau GT, Tajik AJ: Novel risk factors for atherosclerosis. Mayo Clin Proc 2000;75:369–380. Eikelboom JW, Lonn E, Genest J, Jr, Hankey G, Yusuf S: Homocyst(e)ine and cardiovascular disease: a critical review of the epidemiologic evidence. [see comments] Ann Intern Med 1999;131: 363–375. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of increasing folic acid intakes. [see comments] JAMA 1995;274:1049–1057. Moustapha A, Robinson K: High plasma homocysteine: a risk factor for vascular disease in the elderly. Coron Artery Dis 1998; 9:725–730. Kannel WB: Cardiovascular risk factors in the elderly. Coron Artery Dis 1997;8:565–575. Bates CJ, Mansoor MA, van der Pols J, Prentice A, Cole TJ, Finch
15.
16.
17.
18.
19.
397
S: Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over. Eur J Clin Nutr 1997; 51:691– 697. Przygoda P, Janson J, O’Flaherty M, Waisman G, Galarza CR, Alfie J, Camera LA, Camera MI, Mayorga LM: Lack of effective blood pressure control among an elder hypertensive population in Buenos Aires. Am J Hypertens 1998;11:1024–1027. Quintana I, Freeman D, Galarza C, Murua A, Spence JD, Kordich L: Validation of an enzyme immunoassay for the determination of total homocysteine in plasma. [In Process Citation] Blood Coagul Fibrinolysis 2000;11:235–238. Bostom AG, Rosenberg IH, Silbershatz H, Jacques PF, Selhub J, D’Agostino RB, Wilson PW, Wolf PA: Nonfasting plasma total homocysteine levels and stroke incidence in elderly persons: the Framingham Study. Ann Intern Med 1999;131:352–355. Selhub J, Jacques PF, Rosenberg IH, Rogers G, Bowman BA, Gunter EW, Wright JD, Johnson CL: Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991–1994): population reference ranges and contribution of vitamin status to high serum concentrations. [see comments] Ann Intern Med 1999;131:331–339. Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH: Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. [see comments] JAMA 1993;270:2693–2698. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE: Plasma homocysteine levels and mortality in patients with coronary artery disease. [see comments] N Engl J Med 1997; 337:230–236. Snow CF: Laboratory diagnosis of vitamin B12 and folate deficiency: a guide for the primary care physician. [see comments] Arch Intern Med 1999;159:1289–1298. Bots ML, Launer LJ, Lindemans J, Hoes AW, Hofman A, Witteman JC, Koudstaal PJ, Grobbee DE: Homocysteine and short-term risk of myocardial infarction and stroke in the elderly: the Rotterdam Study. Arch Intern Med 1999;159:38– 44. Bostom AG, Silbershatz H, Rosenberg IH, Selhub J, D’Agostino RB, Wolf PA, Jacques PF, Wilson PW: Nonfasting plasma total homocysteine levels and all-cause and cardiovascular disease mortality in elderly Framingham men and women. Arch Intern Med 1999;159:1077–1080. Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrom LE, Ueland PM, Palma-Reis RJ, Boers GH, Sheahan RG, Israelsson B, Uiterwaal CS, Meleady R, McMaster D, Verhoef P, Witteman J, Rubba P, Bellet H, Wautrecht JC, de Valk HW, Sales Luis AC, Parrot-Rouland FM, Tan KS, Higgins I, Garcon D, Andria G, et al: Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project [see comments]. JAMA 1997; 277:1775–1781. Sutton-Tyrrell K, Bostom A, Selhub J, Zeigler-Johnson C: High homocysteine levels are independently related to isolated systolic hypertension in older adults. Circulation 1997;96:1745–1749. Malinow MR, Duell PB, Hess DL, Anderson PH, Kruger WD, Phillipson BE, Gluckman RA, Block PC, Upson BM: Reduction of plasma homocyst(e)ine levels by breakfast cereal fortified with folic acid in patients with coronary heart disease. [see comments] N Engl J Med 1998;338:1009–1015. Staessen JA, Wang JG, Thijs L, Fagard R: Overview of the outcome trials in older patients with isolated systolic hypertension. J Hum Hypertens 1999;13:859– 863.
2002; 15:394 –397
Prevalence of Hyperhomocysteinemia in an Elderly Population Jorge J. Janson, Carlos R. Galarza, Alicia Muru´ a, Irene Quintana, Pablo A. Przygoda, Gabriel Waisman, Luis Camera, Lucia Kordich, Margarita Morales, Luis M. Mayorga, and Mario I. Camera Background: Currently, total hyperhomocysteinemia (tHHcy) is a well-known condition linked to a higher risk of vascular disease. Prevalence of HHcy increases in elderly persons as the risk associated with it persists. Because factors can be potentially reduced in the elderly, it is important to carry out epidemiologic studies of HHcy. Procedure: Previously we described the prevalence of hypertension control in an elder population; now, in an observational cross-sectional simple blind study, total homocysteine (tHcy) concentration was determined in 196 of 400 patients from the original cohort. Results: Mean Hcy concentration was 13.2 mol/L (95% confidence interval 12.4 –14.0; range, 5.0 to 48.9); 15.0 mol/L for men and 12.3 mol/L for women. Mean serum folic acid levels were 4.9 ⫾ 3.1 ng/mL (range, 2.0
S
everal epidemiologic prospective and case-control studies supported the observation that high levels of homocysteine (Hcy) are an important risk factor in arteriosclerotic vascular disease affecting coronary, cerebral, and peripheral arteries.1,2 Risk increases linearly, and each basal tHcy increase of 5 mol/L in fasting subjects is associated to a relative risk of vascular arteriosclerotic disease of 1.35 (95% confidence interval [CI] 1.1–1.6) for men and 1.42 (95% CI 0.99 –2.55) for women.3 Homocysteine levels increase significantly with age, and total hyperhomocysteinemia (tHHcy) prevalence has been reported to be higher in the elderly than in other age groups.4 – 6 However, it is still unknown whether it is due to nutritional causes or is related to metabolic changes common to old age and poor nutritional absorption. The purpose of this study was to evaluate the levels of tHcy in elderly persons living in the city of Buenos Aires, Argentina. Received February 12, 2001. First Decision December 27, 2001. Accepted February 13, 2002. From the Sociedad Argentina de Medicina Vascular (JJJ, CRG, AM, MM, MIC); Facultad de Ciencias Exactas y Naturales, UBA (JJJ, CRG, IQ, LK); and Hospital Italiano de Buenos Aires, Servicio de Clinica 0895-7061/02/$22.00 PII S0895-7061(01)02165-3
to 20.0 ng/mL), and vitamin B12 levels were 384.8 ⫾ 314.1 pg/mL (range, 48.0 to 1500.0 pg/mL). Taking into account the reference values established by the Third National Health and Nutrition Examination Survey III study, HHcy was detected in 69.8% of all the subjects evaluated. The study showed that 76.2% of the men and 66.4% of the women had high Hcy levels. Conclusions: The very high prevalence of tHHcy in the elderly population, and the consequent risks associated with it suggest that although there are no trials that effectively prove the benefit of tHcy decrease, nutritional intervention is still justified. Am J Hypertens 2002;15: 394 –397 © 2002 American Journal of Hypertension, Ltd. Key Words: Homocysteine, elderly people, prevalence, epidemiologic studies.
Methods Subjects An observational, simple blind study was carried out in 202 individuals older than 65 years, from an original sample of 400 patients. These subjects were randomly chosen from a group of 35,800 elderly people from the Unit No. 2 of the National Health Care Program called PAMI (Programa de Atencio´n Me´dica Integral) of the Hospital Italiano de Buenos Aires, who had participated in a previous study.7 Eventually samples from 196 subjects were eligible for Hcy determination. All subjects were evaluated on the basis of a survey of symptoms related to coronary, cerebral, and peripheral diseases. Data were completed with a physical examination, an electrocardiogram (ECG), and a questionnaire on risk factors (smoking, alcohol, stress, sedentary lifestyle). Participants were also asked about the use of vitamin supplementation and drugs, as well as about other causes Medica y Unidad de Hipertension arterial, Buenos Aires, Argentina. Address correspondence and reprint requests to Dr. Jorge J. Janson, Unidad de Hipertension Arterial, Hospital Italiano de Buenos Aires, Gascon 450 (1181), Buenos Aires, Argentina; e-mail: [email protected] © 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
AJH–May 2002–VOL. 15, NO. 5
HYPERHOMOCYSTEINEMIA IN AN ELDERLY POPULATION
of tHHcy. In subjects with previous diagnoses of vascular disease, objective data on these diagnoses were included. Patients with the following pathologies were considered vascular disease carriers. Peripheral arteriopathy: Subjects having undergone aortofemoral or femoropopliteal bypass surgery or with previous intermittent claudication and tests showing a decrease in arterial flow in the lower limbs (Doppler echography, arteriography, or plethysmography). Ischemic cerebral disease: Subjects previously having temporary motor or sensitive focal neurologic deficit, or with sequel and computerized axial cerebral tomography without signs of hemorrhage, and subjects with or without symptoms with Doppler echography of carotid arteries with obstructions higher than 20%. Coronary pathologies: Subjects having undergone myocardial revascularization surgery or angioplasty, or subjects with or without previous thoracic pain and ECG sequel necrosis, or with previous thoracic pain with positive complementary tests for ischemia (ergometry, ventriculogram), and subjects with previous thoracic pain and under antiangina medication with no complementary tests. Informed consent to participate in the study was obtained from all subjects according to guidelines set out by our institutional review board. Blood Samples Blood samples from fasting subjects were obtained using EDTA as anticoagulant. Samples were immediately chilled at ⬍4°C and centrifuged (2000 ⫻ g, 10 min) within 1 h of collection. Plasma was immediately frozen at ⫺70°C until Hcy determination. Serum samples were obtained and stored at ⫺70°C until analysis. Laboratory Determinations Total plasma Hcy levels were determined by enzyme immunoassay (Axis Biochemicals ASA, Oslo, Norway). This method was validated by our research group.8 Serum folic acid and vitamin B12 were determined in the sera of 70 patients by a combined system of competitive immunoassay and chemiluminescence (AccessSanofi-Pasteur; Access Beckman Coulter, Chaseka, MN). This procedure involved the use of monoclonal antibodies, paramagnetic particles, and a chemiluminescence substrate. The light emitted, which was inversely proportional to the concentration of folic acid or vitamin B12, was registered. The study of each patient was completed with the determination of total cholesterol, triglycerides, HDL-cholesterol, and creatininemia (applying the usual laboratory techniques). Statistical Methods Homocysteine, folic acid, and vitamin B12 showed a bias in the distribution of frequencies. Therefore, we performed a logarithmic transformation (base 10) to obtain an adequate normal distribution for the application of parametric
395
Table 1. General characteristics of the population % Women Sedentary Diabetes Widow/widower Married Patients with vascular disease Patients with coronary disease
68.4 64.3 17 33 48.9 17.3 10.2
tests. Thus, when we refer to the mean of these variables, we use the geometric mean (⫾95% CI). Results are expressed as mean ⫾ SD if nothing else is specified. Pearson correlation coefficients and probabilities were calculated for the relationship between HHcy and continual quantitative variables (folic acid, vitamin B12, creatininemia, age, etc.). The analyses of differences between the groups with and without vascular disease were performed by Student t test for two-tailed independent samples. Frequency distribution of nominal variables was analyzed for different values of HHcy applying the Fisher’s exact test and the 2 test. A multivariate analysis (logistic regression) was carried out taking vascular disease as the dependent variable, and different known risk factors plus Hcy as the continual and dichotomic variable with two different values (12 to 15 mol/L).
Results In the group studied (62 men and 134 women), mean age for the patients was 74.4 years (range, 66 to 93 years). General characteristics of the population are shown in Tables 1 and 2. Total plasma tHcy concentration was measured in 196 subjects from the group already described. Mean concentration was 13.2 mol/L (95% CI 12.4 –14.0; range, 5.0 to 48.9 mol/L), 15.0 mol/L (95% CI 13.6 –16.7; fifth percentile 7.0 mol/L and 95th percentile 30 mol/L) for men, and 12.3 mol/L (95% CI 11.5–13.2; fifth percentile 7.2 mol/L and 95th percentile 21.5 mol/L) for women. As it was expected, the differences in total Hcy between sexes were statistically significant (P ⬍ .05). Homocysteinemia correlated weakly with age (r ⫽
Table 2. Clinical characteristics of the population Media (SD) 2
BMI (kg/m ) Creatinine (mg/dL) Cholesterol (total) (mg/dL) HDL (mg/dL) Tg (mg/dL)
27.31 1.05 234.14 48.79 125.23
(4.74) (0.18) (39.86) (7.77) (60.6)
BMI ⫽ body mass index; HDL ⫽ high-density lipoprotein; Tg ⫽ triglyceride.
396
HYPERHOMOCYSTEINEMIA IN AN ELDERLY POPULATION
0.195), creatinine (r ⫽ 0.304), body mass index (r ⫽ 0.221), and folic acid (r ⫽ ⫺0.435). In 34 patients, previous obstructive vascular disease was detected (20 coronary disease, 11 peripheral arteriopathy, 10 previous ischemic cerebrovascular disease, and 3 carotid disease). Ten of the 34 patients had symptomatic atheromatose disease in more than one location. From these, 45% had at least another vascular disease risk factor, apart from increased levels of Hcy. Mean tHHcy in vascular patients (14.4 mol/L; 95% CI 12.6 –16.5) was 14.2% higher than in nonvascular subjects (12.7 mol/L; CI 95% 11.8 –13.6). This difference, however, was not significant. Vitamin Levels Vitamins were measured in sera from 70 patients chosen at random. Serum folic acid level was 4.9 ⫾ 3.1 ng/mL (range, 2 to 20 ng/mL) and vitamin B12 level was 384.8 ⫾ 314.1 pg/mL (range, 48 to 1500 pg/mL). Low levels of vitamin B12 (⬍180 pg/mL) were observed in 14.7% of the subjects, and low levels of plasma folic acid (⬍3 ng/mL), in 24.7% of the subjects, independently of tHHcy. Among individuals with high tHcy levels, deficits of vitamin B12 and folic acid were 15.38% and 32.7%, respectively. Conversely, 94% (n ⫽ 17) of the subjects with low folic acid, and 90% (n ⫽ 9) of the subjects with vitamin B12 deficit showed high tHcy levels.
Discussion Because the association between tHHcy and risk of vascular events is gradual and continual, and tHcy levels are highly prevalent,9 it is still difficult to establish reference values. Recently, the Third National Health and Nutrition Examination Survey (NHANES III) study10 established that higher values (95th percentile) in a healthy, young population (aged 20 to 39 years) with an adequate vitamin plasma level and without renal insufficiency can be considered having tHHcy. On the basis of these criteria, tHHcy was defined for values higher than 11.4 mol/L in men and 10.4 mol/L in women. Taking into account the values considered normal for the American group evaluated by the NHANES III study, our study showed a very high prevalence of tHHcy in a randomly chosen group of elderly people living in the city of Buenos Aires. Thus, 69.8% of the subjects were considered to have tHHcy. The proportion of tHHcy in men (76.2%) was significantly higher than in women (66.4%). On the other hand, tHHcy prevalence in American individuals older than 60 years old was 43.2% and 46.5% for men and women, respectively. Several studies reported that plasma tHcy levels increase with age, depending on nutritional and metabolic factors, and on changes in vitamin B12 absorption and renal excretion. In our country, eating habits are characterized by high consumption of animal proteins and low ingestion of food
AJH–May 2002–VOL. 15, NO. 5
rich in folates. In addition, there is a high prevalence of the thermolabile variant of the methylene tetrahydrofolate reductase in our population. In a cohort of elderly survivors from the original population of Framingham, mean tHcy concentration was 11.9 mol/L, values higher than 14 mol/L (90th percentile in this population) were detected in 29.3% of the subjects.11 On the basis of these criteria, our study reported 44.5% of elderly patients with high Hcy levels. On the other hand, values ⱖ9 mol/L, which was the lowest mortality level in the group studied by Nygard et al,12 were found in 79% of our cases. Subjects defined as hyperhomocysteinemic (NHANES III criteria) in our study also showed high prevalence of vitamin deficit, 15.38% for vitamin B12 and 32.7% for folic acid. Although the relative importance of vitamin deficits to determine Hcy level decreases with age (population attributable risk), it has been reported that between one third and two thirds of elderly people with high tHcy levels also have vitamin deficits. Folic acid deficit is considered the essential cause of high tHcy concentration in young people, whereas the B12 deficit increases with aging.13 Consequently, the treatment of tHHcy in elderly people requires an increase in the consumption of food with high content of folates and vitamin B12. Moreover, if pharmacologic supplementation is added, it would be advisable to measure vitamin B12 or administer orally high doses of B12 together with folic acid (approximately 1% is absorbed in cases of atrophic gastritis). Assuming that no selection bias was present in our study, the probability of obtaining an “ideal” median, 9 mol/L, is less than one in a million (z ⫽ ⫺12.81). As in the case of other cardiovascular risk factors such as cholesterol or arterial hypertension, the relative risk added by tHcy decreases with age. In the past, this led to the underestimation of the impact of risk factors in elderly people, with the consequent disregard for their treatment. This situation has been reversed, not only understanding the significant differences between relative and absolute risks, but also gathering evidence to demonstrate that the association between the factor (ie, tHcy) and the vascular event continues with age. In the Rotterdam study,14 analyses carried out on a cohort of 7983 residents, all older than 55 years at the beginning of treatment, showed increases between 5% and 10% for the occurrence of different vascular events per each 1 mol/L tHcy increase. In a 4-year follow-up, participants of the upper quintile with levels higher than 18 mol/L showed a significant increase of myocardial infarction risk (odds ratio, 2.4; CI 95% 1.1–5.4) and cerebrovascular accident (odds ratio 2.5; CI 95% 1.2–5.4) when compared with those from the lower quintile with values less than 12.0 mol/L. In a recent analysis on 10-year mortality in elderly people in Framingham (n ⫽ 1933), values of tHcy higher than 14.3 mol/L were associated to a nonadjusted rela-
AJH–May 2002–VOL. 15, NO. 5
HYPERHOMOCYSTEINEMIA IN AN ELDERLY POPULATION
tive risk of cardiovascular death of 2.2 (95% CI 1.7–2.8) in respect of lower values.10,15 The European Concert Project studied the interaction between tHcy and other cardiovascular risk factors, finding important implications for risk management. This study deserves special consideration for the interaction between tHcy and hypertension, and tHcy and smoking, because they are more than multiplicative. The study reported an odds ratio of 11 for the interaction between fasting tHcy and hypertension and an odds ratio of 4.6 for fasting tHcy and smoking.16 The subjects from our study showed, in addition to the high tHHcy prevalence (69.8%), a high proportion of cases with arterial hypertension (77.5%). Eight percent of our subjects were smokers. This is a group with high global vascular risk. From other point of view, tHcy might be a causal factor for elevated blood pressure.17 Therefore, according to previous data, just improving eating habits in elderly people could bring significant benefits.18 However, it is important that simultaneous prospective intervention studies were carried out to evaluate the impact of tHcy decrease in the morbidity and mortality due to atherothrombotic disease. Reports on these data are particularly important in the case of elderly people with high absolute risk of vascular disease, in whom intervention studies on other risk factors proved successful.19
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgments The following investigators participated and are gratefully acknowledged: Martin O’Flaherty, Carolina Ponce de Leon, Leonardo Garfi, Oscar Lopez, and Alejandro Manzur.
References 1. 2.
3.
4.
5. 6.
Kullo IJ, Gau GT, Tajik AJ: Novel risk factors for atherosclerosis. Mayo Clin Proc 2000;75:369–380. Eikelboom JW, Lonn E, Genest J, Jr, Hankey G, Yusuf S: Homocyst(e)ine and cardiovascular disease: a critical review of the epidemiologic evidence. [see comments] Ann Intern Med 1999;131: 363–375. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of increasing folic acid intakes. [see comments] JAMA 1995;274:1049–1057. Moustapha A, Robinson K: High plasma homocysteine: a risk factor for vascular disease in the elderly. Coron Artery Dis 1998; 9:725–730. Kannel WB: Cardiovascular risk factors in the elderly. Coron Artery Dis 1997;8:565–575. Bates CJ, Mansoor MA, van der Pols J, Prentice A, Cole TJ, Finch
15.
16.
17.
18.
19.
397
S: Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over. Eur J Clin Nutr 1997; 51:691– 697. Przygoda P, Janson J, O’Flaherty M, Waisman G, Galarza CR, Alfie J, Camera LA, Camera MI, Mayorga LM: Lack of effective blood pressure control among an elder hypertensive population in Buenos Aires. Am J Hypertens 1998;11:1024–1027. Quintana I, Freeman D, Galarza C, Murua A, Spence JD, Kordich L: Validation of an enzyme immunoassay for the determination of total homocysteine in plasma. [In Process Citation] Blood Coagul Fibrinolysis 2000;11:235–238. Bostom AG, Rosenberg IH, Silbershatz H, Jacques PF, Selhub J, D’Agostino RB, Wilson PW, Wolf PA: Nonfasting plasma total homocysteine levels and stroke incidence in elderly persons: the Framingham Study. Ann Intern Med 1999;131:352–355. Selhub J, Jacques PF, Rosenberg IH, Rogers G, Bowman BA, Gunter EW, Wright JD, Johnson CL: Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991–1994): population reference ranges and contribution of vitamin status to high serum concentrations. [see comments] Ann Intern Med 1999;131:331–339. Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH: Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. [see comments] JAMA 1993;270:2693–2698. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE: Plasma homocysteine levels and mortality in patients with coronary artery disease. [see comments] N Engl J Med 1997; 337:230–236. Snow CF: Laboratory diagnosis of vitamin B12 and folate deficiency: a guide for the primary care physician. [see comments] Arch Intern Med 1999;159:1289–1298. Bots ML, Launer LJ, Lindemans J, Hoes AW, Hofman A, Witteman JC, Koudstaal PJ, Grobbee DE: Homocysteine and short-term risk of myocardial infarction and stroke in the elderly: the Rotterdam Study. Arch Intern Med 1999;159:38– 44. Bostom AG, Silbershatz H, Rosenberg IH, Selhub J, D’Agostino RB, Wolf PA, Jacques PF, Wilson PW: Nonfasting plasma total homocysteine levels and all-cause and cardiovascular disease mortality in elderly Framingham men and women. Arch Intern Med 1999;159:1077–1080. Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrom LE, Ueland PM, Palma-Reis RJ, Boers GH, Sheahan RG, Israelsson B, Uiterwaal CS, Meleady R, McMaster D, Verhoef P, Witteman J, Rubba P, Bellet H, Wautrecht JC, de Valk HW, Sales Luis AC, Parrot-Rouland FM, Tan KS, Higgins I, Garcon D, Andria G, et al: Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project [see comments]. JAMA 1997; 277:1775–1781. Sutton-Tyrrell K, Bostom A, Selhub J, Zeigler-Johnson C: High homocysteine levels are independently related to isolated systolic hypertension in older adults. Circulation 1997;96:1745–1749. Malinow MR, Duell PB, Hess DL, Anderson PH, Kruger WD, Phillipson BE, Gluckman RA, Block PC, Upson BM: Reduction of plasma homocyst(e)ine levels by breakfast cereal fortified with folic acid in patients with coronary heart disease. [see comments] N Engl J Med 1998;338:1009–1015. Staessen JA, Wang JG, Thijs L, Fagard R: Overview of the outcome trials in older patients with isolated systolic hypertension. J Hum Hypertens 1999;13:859– 863.