Getting to the heart of homocysteine testing
In 1991, the Mayo Clinic performed a respectable number of
homocysteine analyses-about 800. In 1998, the Rochester, Minn., facility
completed an extraordinary number of homocysteine tests, upwards of
45,000, and in 2001, Mayo Clinic expects the total to increase another
The volume of homocysteine testing has grown steadily
since the early ’90s when prospective trials and case-controlled
studies started showing that this amino acid product of methionine
demethylation was an independent risk factor for cardiovascular
disease. "It’s pretty clear from most cross-sectional and case-controlled
studies and some prospective studies that hyperhomocysteinemia increases
the risk for not only myocardial infarction, but also coronary artery
disease, peripheral vascular disease, stroke, cerebrovascular disease,
restenosis of coronary arteries that have undergone balloon angioplasty,
and even death from coronary artery disease," says Herbert Naito,
PhD, chief, ancillary testing and satellite facilities, Louis Stokes
Cleveland VA Medical Center (Arch Intern Med. 2000;160:422-434).
Now that laboratory methods make it comparatively easy to measure
homocysteine, physicians are jumping on the homocysteine bandwagon-even
though there has been no clear clinical proof that a decrease in
homocysteine will protect an individual from a future cardiovascular
But clinicians are faced with many unanswered questions: Should
homocysteine be used to screen general patient populations or only
those predisposed to cardiovascular risk? Will homocysteine become
another cholesterol? How should clinicians use the results of homocysteine
testing to counsel patients?
Laboratory professionals, meanwhile, are wrestling with questions
of their own: What is a normal homocysteine level? How do homocysteine
tests compare in their analytical precision and bias? Is total homocysteine
equal to reduced and oxidized homocysteine?
At the American Association for Clinical Chemistry meeting
last July, Joseph McConnell, PhD, explained that in vitro studies
suggest a causal relationship between elevated homocysteine and
cardiovascular disease. Homocysteine oxidizes low-density lipoprotein,
which may precipitate atherosclerosis and is toxic to the endothelium.
Homocysteine also decreases the expression of thrombomodulin on
the surface of endothelial cells. Thrombomodulin interacts with
the protein C activation pathway, and its decrease results in inhibition
of this anticoagulant pathway, potentially promoting thrombosis.
Homocysteine activates platelets and increases platelet aggregation,
says Dr. McConnell, director of the cardiovascular risk assessment
laboratory and codirector of the biochemistry genetics laboratory
at Mayo. Dr. McConnell adds that most in vitro studies have been
performed at much higher than physiologic homocysteine concentrations,
so the suggested causal relationship between homocysteine and cardiovascular
disease implied from these studies should be viewed with a grain
In addition to causing cardiovascular disease by increasing thrombogenic
events, hyperhomocysteinemia may trigger atherosclerotic events
by enhancing smooth muscle cell proliferation, intimal-medial wall
thickness, endothelial cell injury, thromboxane A2 formation, lipid
abnormalities, LDL oxidation, and lipoprotein (a) binding to fibrin,
says Dr. Naito (Ann Intern Med. 1999;131:363-375).
Clinical trials are beginning to suggest that lowering the homocysteine
level is beneficial. A mild to moderate elevation in homocysteine
can increase the risk of coronary artery disease by 2.5 times. Projections
from a number of small studies suggest that lowering homocysteine
by only 5 µmol/L may reduce the death rate from coronary artery
disease by 10 percent, Dr. Naito says (JAMA. 1995;274:1049-1057).
A drop in the plasma homocysteine concentration also may cut the
rate of recurrence of restenosis of blood vessels. "So there’s enough
evidence that hyperhomocysteinemia is an independent and dose-related
risk factor, and it may even be a causal factor. It may be like
glucose in that if it gets too high, it becomes a real toxin," Dr.
But how much homocysteine is too much? Most laboratories use 15
µmol/L as the cutoff point between normal and abnormal. A study
conducted from 1991 to 1994 and published in the Annals of Internal
Medicine pointed out, however, that the reference range may
be much lower. This study is unique, says Dr. Naito, because the
authors carefully selected their reference population to exclude
those with established risk factors for hyperhomocysteinemia-that
is, those with low vitamin B12 or folate intake, persons with renal
dysfunction, and pregnant or postmenopausal women. According to
the data, the reference range for men appears to be between 5 and
11.4 µmol/L; in women it ranges from 4 to 10.4 µmol/L. A finding
above 11.4 µmol/L in men and 10.4 µmol/L in women can be considered
abnormal (Ann Intern Med. 1999;131:331-339).
Determining the normal range for homocysteine has been complicated
in the last few years by the addition of folic acid to bread and cereal
products. Because folate fortification changes the normal reference
values for homocysteine, it interferes with prospective studies of
risk reduction and cardiovascular disease event outcomes, said Dr.
McConnell in an interview with CAPTODAY. In the middle of these studies,
he points out, the homocysteine level changed for the entire population-not
just the patients taking vitamins.
Larry Brace, PhD, associate professor of pathology and director
of coagulation services, University of Illinois, Chicago, adds that
a direct relationship may exist between the risk of atherosclerosis
and incrementally rising homocysteine levels. "If you could reduce
your serum homocysteine level to near zero, that would present the
lowest risk for atherosclerosis," he says. "But any incremental
rise above that may carry some risk, even for someone with homocysteine
in the normal range."
To think that homocysteine may be similar to cholesterol is tantalizing.
In addition to two major clinical trials with hard cardiovascular
endpoints published by 1988, as well as other trials examining angiographic
evidence of coronary arteries, an explosion of more recent evidence
confirms the relationship between cholesterol and coronary artery
disease. "That is just not the case with homocysteine," says Andrew
G. Bostom, MD, laboratory director, Memorial Hospital of Rhode Island,
Pawtucket. "I’m not saying we won’t have those data, it’s just that
we don’t have the data now, and it will probably be another five
years to have comparable evidence to what we used to base the 1988
cholesterol education panel guidelines on."
As was done for cholesterol, a panel of experts would have to
examine an abundance of data to decide what level represents a risk
factor for CAD, Dr. Naito says. "How do we get a reliable figure
that we can depend on?" he asks. "What are the variables, besides
aging, gender, adequate vitamin B6, B12, folate intake, disease
states, and medication interference, that can influence homocysteine
concentration in the blood? In addition, what is the biological
variation of homocysteine in the blood from day to day? What is
the best method of treatment? And how do we know that if someone
is below a certain level, they are at minimal risk?
"Another question is, should we screen the general public for
hyperhomocysteinemia?" asks Dr. Naito. "Perhaps clinicians should
agree that homocysteine should be analyzed in only target patient
groups. Patients who have none of the conventional risk factors
for cardiovascular disease but have clinical evidence of atherosclerosis
are ideal candidates," he says. Other good candidates are patients
who have a family history of premature coronary artery disease or
early death due to heart disease and patients who have balloon angioplasty
or carotid artery bypass grafts. Homocysteine levels are higher
in older patients, particularly men, though women catch up after
menopause (Atherosclerosis. 2000;149:163-168).
Diabetics are at greater risk for elevated homocysteine and cardiovascular
consequences; just a 5-µmol/L increase in homocysteine has been
associated with a 60 percent increase in cardiovascular disease
mortality among diabetics, as compared to a 17 percent increase
in those without diabetes mellitus (Circulation. 2000;101:
1506-1511). Homocysteine also is higher in patients with hyperthyroidism,
says Dr. Naito (Ann Intern Med. 1999;131:348-351).
A recent study in Circulation (2000;102:605-610) suggested
that if cardiac bypass patients with high baseline homocysteine
levels are not treated within 2.5 years, they have a 2.6 times greater
risk of recurring cardiac events, adds Dr. Naito. However, other
evidence of a direct connection between a reduction in homocysteine
and a drop in cardiovascular events is lacking, Dr. McConnell says.
So what should laboratory professionals tell clinicians who are
ordering homocysteine tests for their patients? "Physicians in local
hospitals who are potentially going to be using this methodology
should be notified that homocysteine is a marker for cardiovascular
disease, but efforts to reduce homocysteine may not affect patients’
risk for cardiovascular events," Dr. McConnell says.
Patients who already have other cardiovascular disease risk factors
may benefit from knowing they have elevated homocysteine so they
can be more aggressive about reducing their LDL cholesterol or maintaining
a heart healthy diet, he adds.
For laboratory professionals, the question is which homocysteine
test to use. Until recently, high-performance liquid chromatography
was the most common method, and two HPLC methods came to the forefront,
the primary difference being the fluorescent derivatizing agent used.
Each method has its advantages and disadvantages. Monobromobimane
derivatization occurs fairly rapidly, so it lends itself to automated
analysis, Dr. McConnell says. Sodium borohydride typically is used
to reduce homocysteine bound to protein or other thiols to produce
free homocysteine. Derivatization with monobromobimane then occurs
and takes only a few minutes. Monobromobimane obtained from commercial
sources often has fluorescent impurities, which necessitates gradient
elution to separate all other peaks from homocysteine, the peak of
HPLC methods that use SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) or ABD-F (4 aminosulfonyl-7-fluoro-benzo-2-oxa -1,3-diazole) are not as easily adapted to automation, Dr. McConnell
says. Reduction typically is done with tris-carboxethyl-phosphine
(TCEP) and takes about 30 minutes. Proteins are precipitated with
trichloroacetic acid. Derivatization takes a little longer with
SBD-F-about 60 minutes at 60°C-than with monobromobimane, but SBD-F
has fewer fluorescent impurities, "which allows for efficient separation
of homocysteine from other compounds using isocratic HPLC elution
methods," Dr. McConnell says. "Chromatographic times are typically
less than five minutes," he adds.
Subject to debate is that some HPLC methods use internal standards,
but others do not. "When choosing an internal standard, you would
ideally want a substance that is not present in the body but that
has characteristics similar to homocysteine," Dr. McConnell says.
"There is always a question as to whether a molecule that is not
homocysteine behaves exactly like homocysteine during both the reduction
and derivatization processes." Of all the internal standards that
have been used, he adds, cystamine appears to be becoming the internal
standard of choice.
Also a problem for many laboratories is that they do not have
the equipment, technical expertise, or time to adopt HPLC technology,
says Christine Pfeiffer, PhD, chief, global micronutrient section,
National Center for Environmental Health at the Centers for Disease
Control and Prevention, Atlanta. (Drew Scientific, Portsmouth, RI,
however, recently introduced an HPLC system, DS30, that does not
require special technical expertise.)
Providing an alternative to HPLC methods are enzyme immunoassay
kits. EIA techniques, which are commercially available in microtiter
plate or automated formats, are based on the same principle: reduction
of homocysteine and mixed disulfides, conversion of free homocysteine
to S-adenosyl-L-homocysteine (SAH), and a competitive immunoassay-enzyme-linked
immunosorbent assay or fluorescence polarization immunoassay for
The fully automated FPIA from Abbott Laboratories, Abbott Park,
Ill., reduces disulfide forms of homocysteine with dithiothreitol,
enzymatically converts homocysteine to SAH, and adds an anti-SAH
antibody and a fluorescein tracer.
Use of such immunoassays is growing. In a CAP Survey of 289 laboratories
that are conducting homocysteine analyses, the first such Survey
conducted by the College, 80 percent were using the technique. FPIA
compares favorably with HPLC.
Good agreement was noted among all laboratories in their analysis
of three samples with varying concentrations of homocysteine, which
were sent out by the College. The coefficient of variation was under
10 percent in most cases, reports Anthony A. Killeen, MD, associate
director for clinical laboratories, University of Michigan Medical
Center, Ann Arbor, and a member of the CAP Clinical Chemistry Resource
Committee. Although the information comes from only one mailing, Dr.
Killeen says, "The initial data look quite promising in terms of analytical
performance of the methods that are in use by laboratories." Data
from a second Survey will be available and analyzed soon, he adds.
Dr. Pfeiffer also found good comparability among HPLC, gas chromatography-mass
spectrometry, and immunoassays. The mean interlaboratory variation
and mean intralaboratory variation was less than 10 percent for
14 government, academic, and clinical laboratories (Clin Chem.
Her analysis and the results of an external quality assessment
program from Denmark published a month later, nevertheless, saw
room for improvement, she says. Although the interlaboratory variation
was low, it might not be acceptable clinically because of the graded
rise in risk for cardiovascular disease with increasing homocysteine,
even within the normal range. Laboratories using the same method
also sometimes varied more among themselves than laboratories that
used different methods.
Dr. Pfeiffer concluded that a need existed to improve analytical
precision so laboratories could use the same reference intervals.
She also determined that it was necessary to evaluate the performance
of individual laboratories with standard reference materials characterized
by a reference method.
"Having standardized reference materials and a reference method
will give the last necessary push to come to closer agreement among
the methods and among laboratories," Dr. Pfeiffer says. "Having
proficiency testing out there helps, because everyone can evaluate
themselves over time and in comparison with other laboratories.
But it’s not the same as having a reference material with a certified
value and a reference method that establishes that value," she adds.
The CDC is optimizing a liquid chromatography-mass spectrometry
technique as a potential reference method, but its development must
be completed and it must be thoroughly evaluated, which will take
several months, Dr. Pfeiffer says.
Scientists from the Mayo Clinic and elsewhere were to meet in
December with representatives from the National Institute of Standards
and Technology to begin discussions regarding preparation of standard
reference materials for homocysteine, Dr. McConnell says. It may
take at least a year before a standard reference material is approved,
But all the proficiency testing in the world isn’t going
to amount to a hill of beans if laboratories don’t pay attention
to preanalytical variables. "The samples that are sent out by the
College have stable levels of homocysteine and thus do not control
for preanalytical variables," says Dr. McConnell. "If you are not
taking care of preanalytical variables, you may be reporting inaccurate
results despite acceptable proficiency testing results."
For laboratories that are beginning to develop homocysteine testing,
control of preanalytical variables is crucial, Dr. McConnell adds,
because homocysteine in whole blood is highly unstable. If a sample
of blood is collected and left at room temperature, within an hour
the homocysteine level can increase by 10 percent or more, he says.
If left at room temperature for four to 24 hours, homocysteine levels
can rise in whole blood samples by 35 to 75 percent. Samples, therefore,
should be processed within an hour if they aren’t placed on ice,
and they should be centrifuged within 15 minutes if at all possible,
Dr. McConnell advises.
Standard operating procedure in many facilities, however, is for
phlebotomists to collect eight or 10 samples at once. If a sample
for homocysteine testing is first on the list, it may take an hour
or more before it reaches the laboratory. Then it may sit on a bench
for 30 to 45 minutes before a technologist places it in the centrifuge
and spins it down.
Dr. McConnell recommends that laboratory professionals ask phlebotomists
to place samples for homocysteine testing in ice immediately after
they are drawn. If kept on ice at 2°C, homocysteine will not change
significantly for about four hours.
Reports about the need to obtain fasting samples vary. One study
involving only 13 patients found no difference in homocysteine levels
among fasting and nonfasting patients. A second study showed that
homocysteine levels were lower if samples were drawn two to four
hours after breakfast; a third indicated that homocysteine levels
increased six hours after a meal, Dr. McConnell says.
In the experience of Robert H. Williams, PhD, fasting is essential.
Changes in homocysteine levels admittedly are transient after meals;
eating initially will raise the methionine level and eventually alter
homocysteine. The nature of a meal also influences homocysteine levels.
"If you’re going to have eggs and bacon for breakfast, your methionine
levels are going to be sky high. So if you’re trying to get a true
baseline homocysteine level in a patient, you need to know they’ve
been fasting," says Dr. Williams, director of general chemistry and
toxicology and assistant professor of pathology, University of Illinois,
New tests for homocysteine may soon be available to laboratories.
Already at the Mayo Clinic, tandem mass spectrometry is used to
measure homocysteine. The technique, which was developed at Mayo
by Piero Rinaldo, MD, PhD, does not require derivatization. It takes
a stable isotope of homocystine (homocysteine-homocysteine disulfide)
and runs it as the internal standard. "The internal standard thus
controls for differences in reduction as well as any other potential
loss of homocysteine during sample preparation," Dr. McConnell says.
Other, more direct enzymatic-type methods are becoming available,
but Dr. McConnell cautions laboratory professionals to make sure
the methods have been fully validated and compared to established
methods before bringing them onboard.
Also on the horizon is measurement of the multiple forms of homocysteine.
The medical community doesn’t know precisely what each of the various
forms of homocysteine does, says Dr. Williams, who, along with Dr.
Brace, has been studying homocysteine in stroke patients. Drs. Brace
and Williams have been examining the multiple forms of homocysteine
because, "Most compounds, when they are bound to protein, are not
biologically active, so the protein-bound portion of homocysteine
probably is not responsible for any pathological effects," Dr. Williams
More than likely, the reduced form of homocysteine is the most
active form of the compound because the sulfhydryl group is participating
in the reactions, Dr. Williams explains. Still, trying to target
the multiple forms of homocysteine has been difficult because these
substances change quickly in blood after it has been removed from
a patient. Measuring the multiple forms of homocysteine consequently
is time-consuming and ineffective. It requires collecting blood
in a heparinized tube that contains a derivatization agent or a
specific trapping reagent to separate the different forms of homocysteine.
This method traps and measures the reduced or oxidized free fractions
of homocysteine, like homocystine and the mixed disulfide cysteine-homocysteine.
Alternatively, it involves collecting the sample in EDTA, chilling
it immediately, rapidly spinning it down, and separating and acidifying
the plasma-all within 15 minutes or less. "Most laboratories can’t
do that realistically; it just doesn’t happen in the real world,"
Dr. Williams says.
But Dr. Williams has devised another way to test homocysteine
fractions that is less labor intensive. His method is described
in a paper that he has submitted to Clinical Chemistry. For now,
therefore, Dr. Williams cannot elaborate. "Stay tuned for further
developments," he says.
Karen Sandrick is a freelance writer in Chicago.