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(Stroke. 1999;30:1554-1560.)
© 1999 American Heart Association, Inc.

Original Contributions

Homocyst(e)ine and Risk of Cerebral Infarction in a Biracial Population

The Stroke Prevention in Young Women Study

Steven J. Kittner, MD, MPH; Wayne H. Giles, MD, MS; Richard F. Macko, MD; J. Richard Hebel, PhD; Marcella A. Wozniak, MD, PhD; Robert J. Wityk, MD; Paul D. Stolley, MD, MPH; Barney J. Stern, MD; Michael A. Sloan, MD; Roger Sherwin, MB, BChir; Thomas R. Price, MD; Robert J. McCarter, ScD; Constance J. Johnson, MD; Christopher J. Earley, MD, PhD; David W. Buchholz, MD M. Rene Malinow, MD

From the Departments of Neurology (S.J.K, R.F.M., M.A.W.) and Epidemiology and Preventive Medicine (S.J.K., J.R.H., R.J.M., T.R.P., P.D.S.), University of Maryland at Baltimore; Geriatrics Research, Education, and Clinical Center, Baltimore Department of Veterans Affairs Medical Center (Md) (S.J.K., R.F.M); Department of Neurology, Johns Hopkins University, Baltimore, Md (D.W.B., C.J.E., C.J.J., R.J.W.); Division of Adult and Community Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, Ga (W.H.G.); Department of Neurology, Emory University, Atlanta, Ga (B.J.S.); Department of Neuroscience, Harbin Clinic, Rome, Ga (M.A.S.); Tulane School of Public Health and Tropical Medicine, New Orleans, La (R.S.); and Laboratory of Cardiovascular Diseases, Oregon Primate Research Center, Beaverton (M.R.M.).

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion Appendix 1 References

 
Background and Purpose—Genetic enzyme variation and vitamin intake are important determinants of blood homocyst(e)ine levels. The prevalence of common genetic polymorphisms influencing homocyst(e)ine levels varies by race, and vitamin intake varies by socioeconomic status. Therefore, we examined the effect of vitamin intake, race, and socioeconomic status on the association of homocyst(e)ine with stroke risk.

Methods—All 59 hospitals in the greater Baltimore-Washington area participated in a population-based case-control study of stroke in young women. One hundred sixty-seven cases of first ischemic stroke among women aged 15 to 44 years were compared with 328 controls identified by random-digit dialing from the same region. Risk factor data were collected by standardized interview and nonfasting phlebotomy. Plasma homocyst(e)ine was measured by high-performance liquid chromatography and electrochemical detection.

Results—Blacks and whites did not differ in median homocyst(e)ine levels, nor did race modify the association between homocyst(e)ine and stroke. After adjustment for cigarettes per day, poverty status, and regular vitamin use, a plasma homocyst(e)ine level of 7.3 µmol/L was associated with an odds ratio for stroke of 1.6 (95% CI, 1.1 to 2.5).

Conclusions—The association between elevated homocyst(e)ine and stroke was independent not only of traditional vascular risk factors but also of vitamin use and poverty status. The degree of homocyst(e)ine elevation associated with an increased stroke risk in young women is lower than that previously reported for middle-aged men and the elderly and was highly prevalent, being present in one third of the control group.

Key Words: case-control studies • cerebrovascular disorders • homocysteine • risk factors • vitamins

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion Appendix 1 References

 
Homocyst(e)ine is produced from the essential amino acid methionine and metabolized via 3 pathways. It is catabolized to cysteine via the B₆-dependent enzyme cystathionine ß-synthase, remethylated to methionine via the folic acid and B₁₂-dependent enzyme methionine synthase, or remethylated via the enzyme betaine homocysteine methyltransferase.¹ Thus, blood levels of homocyst(e)ine can be affected by genetic enzyme variation or vitamin intake; both factors are known to be important determinants of homocyst(e)ine levels in the population.^{2 3}

The homocyst(e)ine theory of atherosclerosis was first promulgated by McCully⁴ on the basis of a "natural experiment." He observed pathological findings in an infant with a rare inborn error of B₁₂ metabolism similar to those in infants with cystathionine ß-synthase deficiency. He concluded that extreme elevation in blood homocyst(e)ine, the only metabolic abnormality shared by these disorders, was the cause of the vascular disease in these children. Subsequently, a large body of observational epidemiological evidence, primarily case-control studies, linked even mild to moderate elevations in plasma homocyst(e)ine to vascular disease, including stroke, first among young adults and subsequently among persons of all ages.^{5 6}

Nevertheless, relatively few population-based studies of the relationship between homocyst(e)ine and stroke risk have been conducted in representative American populations that include minority groups. This is relevant for 2 reasons. First, vitamin intake varies with socioeconomic status. Second, the prevalence of common genetic polymorphisms, which result in impaired homocyst(e)ine metabolism, varies with race/ethnicity. Since low folic acid and vitamin B₆ intake is common among persons of lower socioeconomic status, including many blacks,^{7 8} it is expected that moderate hyperhomocyst(e)inemia on a nutritional basis would be more prevalent among blacks. In contrast, the most common genetic polymorphism predisposing to elevated homocyst(e)ine is much more common among whites. The homozygous form of the Val MTHFR polymorphism⁹ is associated with moderate hyperhomocyst(e)inemia when folic acid levels are low,³ and its prevalence is estimated at 5% to 10% among whites and 1% among blacks.^{10 11} By affecting the variation of homocyst(e)ine levels in different populations, these factors could influence the observed relationship of homocyst(e)ine to vascular disease events. Therefore, in the biracial population of the Baltimore-Washington corridor, we conducted a case-control study to examine the effect of vitamin intake, race, and socioeconomic status on the association of plasma homocyst(e)ine with stroke risk.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion Appendix 1 References

 
The Stroke Prevention in Young Women Study is a population-based case-control study initiated to study risk factors for ischemic stroke in young women. The term population-based is taken to mean that both cases and their comparison group were identified from the same defined population. The study area included all of Maryland except the far western panhandle, Washington DC, and southern portions of both Pennsylvania and Delaware. Cases and controls were recruited to the study between February 26, 1992, and January 1, 1996. The study was approved by an institutional review committee, and the subjects gave informed consent.

Cases were women aged 15 to 44 years with a first cerebral infarction, identified by discharge surveillance at all 59 hospitals in the study area and through direct referral by regional neurologists. The methods for discharge surveillance, chart abstraction, case adjudication, and assignment of probable and possible underlying causes have been described previously.^{12 13 14} Recruitment within 1 year of stroke was required for participation. Of 291 cases who were both eligible and identified within the 1-year time window after stroke, 227 could be contacted and agreed to participate; 171 had a blood sample obtained for homocyst(e)ine measurement. Of these, 4 were excluded because the blood sample was not processed within the 6-hour time limit, leaving a final sample of 167 cases.

Controls were women without a history of stroke, frequency-matched by age and geographic region of residence to the cases, identified by random-digit dialing. Of 450 eligible controls, 392 agreed to participate, and 335 had a blood sample obtained for homocyst(e)ine measurement. Of these, 7 were excluded because the blood sample was not processed within the 6-hour time limit, leaving a final sample of 328 controls.

Nonfasting blood samples for homocyst(e)ine were obtained at a median time after stroke of 87 days; 30.4% were obtained within 30 days of stroke. Samples for homocyst(e)ine were drawn into EDTA evacuated tubes (Vacutainer, Becton-Dickinson), immediately placed on ice, and transported to a central processing laboratory, where they were centrifuged at 1520g at 4°C for 15 minutes. Plasma homocyst(e)ine is known to be stable when whole blood is kept on ice for 6 hours before centrifugation.¹⁶ Immediately after centrifugation, the plasma samples were placed in cryogenic vials and frozen at -70°C until they were shipped on dry ice to the Oregon Regional Primate Center for analysis. Total plasma homocyst(e)ine (the sum of free or protein-bound homocysteine, the homocysteinyl moieties of homocystine, and cysteine-homocysteine mixed disulfide) was determined by high-performance liquid chromatography and electrochemical detection as previously described.^{17 18 19} Case and control samples were run batched in a 2:1 ratio, and laboratory personnel were blinded as to case-control status. Assays were run in duplicate, the results were averaged, and the analysis was repeated if duplicates differed 10% from the average. Standard curves were determined daily. The mean within-assay within-pair coefficient of variation (standard deviation, multiplied by 100 and divided by the mean) of this method is 3.2%.

Measured potential confounders and effect modifiers of the association between total plasma homocyst(e)ine and stroke included age, race, educational level, poverty status, hypertension, diabetes mellitus, angina or myocardial infarction, current smoking status, alcohol use, regular vitamin use, body mass index, total cholesterol, and HDL cholesterol. Hypertension, diabetes mellitus, and angina or myocardial infarction were determined by asking the study participant (or her proxy if the participant was unable to answer) if she had ever been told by a physician that she had the condition. Similarly, age, race, educational level, current smoking status, regular multivitamin use, alcohol use, and poverty status were determined by subject or proxy report. Current smokers were classified regarding the average number of cigarettes smoked per day, and regular multivitamin users were classified on the basis of the number of years of regular use during the prior 10 years. Poverty status, determined according to the 1993 Federal Poverty Income Guidelines,²⁰ was based on total family income and the number of household members. The poverty threshold was raised to 200% of that in the federal guidelines because of the high cost of living in Maryland and Washington, DC. Body mass index was based on self-report and calculated as the weight in kilograms divided by the square of the height in meters.

Total cholesterol and HDL cholesterol were measured according to standard practice,^{21 22} and levels were considered high at 240 mg/dL and 35 mg/dL, respectively.

Wilcoxon rank sum tests were used to compare medians, and Fisher's exact tests were used to compare proportions. All probability values were 2-sided, and P<0.05 was considered statistically significant. Adjusted odds ratios (ORs) derived from logistic regression were used to determine whether homocyst(e)ine was associated with an increased risk for stroke after controlling for important confounders. A potential confounder was considered important if adjustment for that factor altered the unadjusted OR by 10%.²³ Effect modification was determined by including an interaction term in the logistic regression model.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion Appendix 1 References

 
There were 167 cases included in the analyses. Those omitted were 60 participating patients for whom blood measurements for homocyst(e)ine were either not available or not processed within the 6-hour time window, as well as 33 eligible cases who refused to participate. Compared with the 167 cases included in the analysis, the 60 participants who did not have a homocyst(e)ine value did not have a statistically significant difference in medical record–derived age, race, hypertension, diabetes mellitus, current smoking status, or adjudicated stroke etiology. Compared with the 167 included cases, the 33 refusals were significantly more likely to be black (76% versus 44%), a current smoker (78% versus 45%), and diabetic (29% versus 13%).

Similarly, the 328 controls included in the analyses were compared with the 64 controls for whom blood measurements for homocyst(e)ine were either not available or not processed within the 6-hour time window, as well as with the 38 refusals for whom a brief telephone interview was available. There were no statistically significant differences in the questionnaire-derived age, race, hypertension, diabetes mellitus, or current smoking status between the groups.

Stroke cases were classified as having a probable, possible, or undetermined etiology, as previously described.^{12 13} Among the 167 stroke patients, 83 (50%) had at least 1 probable cause, 32 (19%) had no probable cause but at least 1 possible cause, and 52 (31%) were indeterminate. Table 1 shows the distribution of probable and possible causes. "Other determined causes" of stroke include hematologic disorders, nonatherosclerotic vasculopathy (eg, vasculitis and dissection), migraine, drug abuse, and stroke associated with the postpartum state.

View this table: [in this window] [in a new window]   Table 1. Etiologies Among Cases With a Probable or Possible Cause of Stroke

Table 2 shows the median homocyst(e)ine level in the control group by selected categorical factors and the univariate relationship of these factors to case-control status. Some factors were associated with both homocyst(e)ine and stroke, while others were associated with only homocyst(e)ine or stroke. In this analysis, current cigarette smoking and total cholesterol were directly associated with both homocyst(e)ine and stroke, while regular vitamin use was inversely associated with both factors. Increased age was directly associated only with increased homocyst(e)ine. However, age was not associated with stroke because cases and controls were frequency matched for age. Factors associated only with increased stroke risk were black race, income below the poverty threshold, hypertension, coronary artery disease, diabetes mellitus, and higher body mass index and HDL cholesterol.

View this table: [in this window] [in a new window]   Table 2. Relationship of Selected Factors to Homocyst(e)ine Levels and Case-Control Status

Table 3 shows the crude OR for stroke by quintile of homocyst(e)ine, with the first quintile serving as the reference category. Results are shown for the entire study population and stratified by race. Overall, there is no increase in stroke risk until the fourth quintile (homocyst(e)ine 7.30 to 9.39 µmol/L) (OR=2.0; 95% CI, 1.1 to 3.6) and no appreciable increased risk at the fifth quintile (OR=2.1; 95% CI, 1.2 to 3.8). This pattern was seen both among blacks and among whites and others. Therefore, to maximize power, subsequent analyses examine the risk of stroke associated with being in the higher 2 quintiles (high-homocyst(e)ine group) compared with the lower 3 quintiles (reference group) and do not stratify by race. Overall, 54% of cases and 33% of controls were in the high-homocyst(e)ine group.

View this table: [in this window] [in a new window]   Table 3. Crude ORs for Stroke of Homocyst(e)ine Quintiles in Entire Population and Stratified by Race

Each covariate was screened for effect modification and confounding of the relationship between homocyst(e)ine and stroke. None of these factors modified the relationship, and only the number of cigarettes smoked per day, poverty status, and regular vitamin use were confounders.

Table 4 shows the results of logistic regression analysis in the total study population. The crude OR of 2.3 (95% CI, 1.6 to 3.4) for the high-homocyst(e)ine group was progressively reduced by the sequential addition to the model of cigarettes per day (OR=2.1; 95% CI, 1.4 to 3.1), poverty status (OR=1.8; 95% CI, 1.2 to 2.8), and lack of regular vitamin use (OR=1.6; 95% CI, 1.1 to 2.5). Nevertheless, even in the fully adjusted model, the OR for the high-homocyst(e)ine group remained statistically significant.

View this table: [in this window] [in a new window]   Table 4. OR for Stroke of the High-Hemocyst(e)ine Group Before and After Adjustment for Confounders

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion Appendix 1 References

 
In a large population-based case-control study among black and white women, we found that the association between moderate hyperhomocyst(e)inemia and stroke was independent not only of traditional vascular risk factors but also of vitamin use and poverty status. The magnitude of the increase in risk associated with elevated homocyst(e)ine (OR=1.6) was similar to that associated with smoking a pack of cigarettes per day (OR=1.9). Elevated homocyst(e)ine, as defined in this study, was highly prevalent, being present in one third of the control group

We found no evidence that the relationship between homocyst(e)ine and stroke is different for blacks than for the remainder of the study population, which was predominantly white. Furthermore, there was no evidence that homocyst(e)ine levels were higher in blacks than in whites (Table 2). In fact, the age-adjusted medians were 6.33 µmol/L for blacks and 6.40 µmol/L for whites (P=0.89). These findings regarding median homocyst(e)ine levels are consistent with national data among middle-aged and elderly persons from the National Health and Nutrition Examination Survey (NHANES).²⁴ In contrast, the Northern Manhattan Stroke Study²⁵ found that elderly blacks had higher homocyst(e)ine levels than whites, even after adjustment for age and other determinants of homocyst(e)ine level.

It is noteworthy that cigarette smoking was the only traditional vascular risk factor that required adjustment in our study. We also found it necessary to adjust for 2 other factors, current multivitamin use and poverty status, which have not been commonly included as potential confounders in other studies.^{26 27} The regular use of multivitamins, which generally contain 0.4 mg of folic acid, 2 mg of B₆, and 6 µg of B₁₂, had a strong inverse association not only with homocyst(e)ine level but also with stroke risk. Poverty status had a weaker association with homocyst(e)ine levels, presumably on a dietary basis, but a strong association with stroke risk. Multivitamin use and poverty status had a strong association with stroke risk not only because of their effect on homocyst(e)ine but also because they are markers of other poorly defined health-affecting behaviors. The relationship of elevated homocyst(e)ine to stroke remained statistically significant, though attenuated in strength, after adjustment for these confounders.

Early prospective studies showed no²⁸ or only an equivocal²⁹ relationship of basal homocysteine to stroke risk. Recently, however, the British Regional Heart Study showed a strong, independent, and graded relationship of homocysteine to stroke risk during a 13-year follow-up study of men aged 40 to 59 years.²⁷ The contrasting results of these earlier cohort studies from the British Regional Heart Study may be due to underlying population differences, to inadequate power, or to differing procedures for collection of blood samples. It is known that homocyst(e)ine leaks out of red blood cells relatively rapidly at room temperature; the British study separated the serum from the blood samples within minutes of venipuncture. Our findings differ from the British results in several respects. Homocyst(e)ine values strongly increase with age and, to a lesser extent, with male sex; thus, the fourth quintile in our study would fall within the first or reference quartile of the British study. We did not observe a dose response; our results were more consistent with a threshold response. Unlike suggestions from the British Regional Heart Study²⁷ and the Atherosclerosis Risk in Communities Study,³⁰ we did not find definite evidence of an increased homocyst(e)ine effect in hypertensive subjects.

A potential criticism of this study is that, compared with cases who refused participation, the participating cases had a smaller proportion of blacks, diabetics, and cigarette smokers. This would tend to underestimate the effect of these factors on stroke risk but is unlikely to have substantially biased the OR for the association between homocyst(e)ine and stroke since race and diabetes were not strongly associated with homocyst(e)ine level and the number of cigarettes currently smoked per day was adjusted for in the analyses.

The present study has several limitations. The primary limitation is the fact that, in a case-control design, homocyst(e)ine levels are measured after the stroke. Thus, we cannot determine whether elevations in homocyst(e)ine were a precursor or a consequence of the stroke. Lindgren and colleagues³¹ showed that homocyst(e)ine levels measured in the acute phase (mean, 2 days) after stroke are significantly lower than levels 1 to 2 years later, while no changes occurred in a control group. Similar findings were noted by Verhoef and coworkers³² in regard to myocardial infarction. Egerton and colleagues³³ found that homocyst(e)ine is 25% lower on the first day after myocardial infarction compared with levels at 7 days or 6 months, changes that were inverse to those of C-reactive protein, an acute phase reactant. The findings from these studies are consistent but have an ambiguous interpretation, since they could be due to an acute decrease in homocyst(e)ine or to a poststroke chronic increase in homocyst(e)ine. Distinguishing between these 2 possibilities would require homocyst(e)ine measurement both before and after such an event. While prospective data show that homocyst(e)ine levels predict stroke in middle-aged men,²⁷ a prospective study design is not feasible in the young adult population because of the low incidence of stroke in this age range.

A second limitation of the study is that serum vitamin levels were not measured. Thus, we cannot determine whether there was an association of vitamin status with stroke and, if so, whether this association is entirely mediated by homocyst(e)ine. There is evidence from case-control^{32 34} and prospective³⁵ data that low vitamin B₆ levels are associated with risk of vascular disease, independent of homocyst(e)ine level.

A third limitation of this study and all observational studies is that all exposures were measured with error. It could be argued³⁶ that inadequate measurement of other cardiovascular risk factors³⁷ or their inadequate control in statistical analyses could lead to bias in favor of finding an association between homocyst(e)ine and vascular disease. Certain important confounders, such as serum creatinine^{27 38} and physical activity,³⁷ were not measured, and other factors, such as cholesterol, were measured subsequent to the event when cases would be more likely to have been treated. Conversely, blood samples for homocyst(e)ine were obtained in the nonfasting state under epidemiological field conditions. Had it been possible to reduce this measurement error or adjust for it, the observed relationships between homocyst(e)ine and stroke may have been stronger.

The potential for a causal relationship between moderate hyperhomocyst(e)inemia and vascular disease suggested by observational studies is bolstered by the biological underpinnings of the association. There is evidence that hyperhomocyst(e)inemia is both atherogenic^{30 39 40} and prothrombotic,^{41 42 43 44 45 46} operating through a variety of potential mechanisms including direct endothelial injury, mitogenic activity of vascular smooth muscle cells, impaired endogenous fibrinolysis, endothelial nitric oxide response, and inhibition of thrombomodulin-dependent protein C activation. Our findings in a young population in which atherosclerotic disease is rarely responsible for ischemic stroke^{14 47} support the notion that homocyst(e)ine elevations are associated with a hypercoagulable state. The recent development of animal models for the pathological⁴⁸ and physiological⁴⁹ effects of hyperhomocyst(e)inemia provides further biological plausibility. Nevertheless, there is an important current of dissenting opinion regarding the importance of homocyst(e)ine elevations in the pathogenesis of vascular disease. This opinion is based primarily on criticisms of the applicability of in vitro studies of the biological effects of homocyst(e)ine and a lack of consistency in the prospective studies of homocyst(e)ine and coronary heart disease.⁵⁰ Even prospective studies do not exclude the possible interpretation that vascular disease causes an increase in homocyst(e)ine levels, unless persons with subclinical vascular disease are excluded from the population at risk.⁵⁰ Intervention studies in animal models and humans are needed to determine which of the potential mechanisms of homocyst(e)ine-associated vascular risk are modifiable by targeted vitamin therapy. Most importantly, recently initiated primary and secondary prevention trials will determine whether lowering homocyst(e)ine with B vitamin interventions will lower risk for vascular disease events.

	Acknowledgments

 
This study was supported by a clinical stroke research center award (NS16332-11) from the National Institute of Neurological Disorders and Stroke. Dr Kittner was supported in part by the Geriatric Research, Education, and Clinical Center at the Baltimore Veterans Affairs Medical Center. We are indebted to Barbara Upson for her outstanding technical assistance and to the following members of the Stroke Prevention in Young Women research team for their dedication: Anne Epstein, Barbara Feeser, James Gardner, Mary Keiser, Ann Maher, Jennifer Rohr, Mary J. Seipp, Susan Snyder, Mary J. Sparks, and Nancy Zappala.

	Footnotes

 
Reprint requests to Dr Steven J. Kittner, Bressler Building, Room 12-006, University of Maryland at Baltimore, 655 W Baltimore St, Baltimore, MD 21201.

	Appendix 1

Top Abstract Introduction Subjects and Methods Results Discussion Appendix 1 References

 
The authors would like to acknowledge the assistance of the following individuals who have sponsored the Stroke Prevention in Young Women Study at their institution: Frank Anderson, MD; Clifford Andrew, MD, PhD; Christopher Bever, MD; Nicholas Buendia, MD; Young Ja Cho, MD; James Christensen, MD; Remzi Demir, MD; Terry Detrich, MD; John Eckholdt, MD; Nirmala Fernback, MD; Jerold Fleishman, MD; Benjamin Frishberg, MD; Stuart Goodman, MD, PhD; Norman Hershkowitz, MD, PhD; Luke Kao, MD, PhD; Mehrullah Khan, MD; Ramesh Khurana, MD; John Kurtzke, MD; William Leahy, MD; William Lightfoote II, MD; Bruce Lobar, MD; Micheal Miller, MD, PhD; Harshad Mody, MBBS; Marvin Mordes, MD; Seth Morgan, MD; Howard Moses, MD; Sivarama Nandipati, MD; Mark Ozer, MD; Roger Packer, MD; Thaddeus Pula, MD; Phillip Pulaski, MD; Naghbushan Rao, MD; Marc Raphaelson, MD; Solomon Robbins, MD; David Satinsky, MD; Elijah Saunders, MD; Micheal Sellman, MD, PhD; Arthur Siebens, MD (deceased); Harold Stevens, MD, PhD; Dean Tippett, MD; Roger Weir, MD; Micheal Weinrich, MD; Richard Weisman, MD; Don Wood, MD (deceased); and Mohammed Yaseen, MD.

In addition, the study could not have been completed without support from the administration and medical records staff at the following institutions: in Maryland: Anne Arundel Medical Center; Atlantic General Hospital; Bon Secours Hospital; Calvert Memorial Hospital; Carroll County General; Church Hospital Corporation; Doctors Community Hospital; Fallston General Hospital; Franklin Square Hospital Center; Frederick Memorial Hospital; The Good Samaritan Hospital of Maryland, Inc; Greater Baltimore Medical Center; Harbor Hospital Center; Hartford Memorial Hospital; Holy Cross Hospital; Johns Hopkins Bayview, Inc; Johns Hopkins Hospital; Howard County General Hospital, Inc; Kennedy Krieger Institute; Kent and Queen Anne Hospital; Laurel Regional Hospital; Liberty Medical Center, Inc; Maryland General Hospital; McCready Memorial Hospital; Memorial Hospital at Easton; Mercy Medical Center; Montebello Rehabilitation Hospital; Montgomery General Hospital; North Arundel Hospital; Northwest Hospital Center; Peninsula Regional Medical Center; Physician's Memorial Hospital, Prince George's Hospital Center; Saint Agnes Hospital; Saint Joseph Hospital; Saint Mary's Hospital; Shady Grove Adventist Hospital; Sinai Hospital of Baltimore; Southern Maryland Hospital Center; Suburban Hospital; The Union Memorial Hospital; University of Maryland Medical System; Department of Veterans Affairs Medical Center in Baltimore; Washington Adventist Hospital; and Washington County Hospital; in Washington DC: Children's National Medical Center; District of Columbia General Hospital; The George Washington University Medical Center; Georgetown University Hospital; Greater Southeast Community Hospital; Hadley Memorial Hospital; Howard University Hospital; National Rehabilitation Hospital; Providence Hospital; Sibley Memorial Hospital; Veterans Affairs Medical Center; and the Washington Hospital Center; in Pennsylvania: Gettysburg Hospital and Hanover General Hospital.

Received February 18, 1999; revision received April 13, 1999; accepted May 3, 1999.

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