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Folate status of rheumatoid arthritis patients receiving long-term low-dose methotrexate therapy.

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The folate status of 29 healthy control subjects,
16 rheumatoid arthritis (RA) patients taking methotrexate (MTX), and 20 RA patients who were not being
treated with MTX was estimated by an assay of the
folate-dependent enzymatic synthesis of serine from
formate and glycine, which is termed the C1 index.
Analysis of variance demonstrated that the specific
activity of the enzyme system in lymphocytes was significantly lower in the MTX-treated group, with an activity
approximately one-half that of the control and the
non-MTX-treated groups. Since the C, index is one of
the first biochemical parameters Found to be different
between MTX-treated and non-MTX-treated groups,
alterations in folate-mediated amino acid metabolism
may be involved in the mechanism of response to MTX
therapy. Use of the C, index may assist in the development of protocols which preserve the efficacy of MTX
therapy while minimizing toxicity.
Methotrexate (MTX), an antimetabolite of folk
acid, is widely used in high doses (>20 mg/m2) in the
treatment of neoplastic diseases (1,2). In high doses,
this drug inhibits dihydrofolate reductase, which depletes the supply of reduced intracellular folate co~____
From the School of Medicine, The University of Alabama
at Birmingham.
Supported in part by USPHS grants CA-28103 and GM23453.
Sarah L. Morgan, MD, RD: Divisions of Clinical Nutrition
and General and Preventive Medicine; Joseph E. Baggott, PhD:
Division of Nutritional Biochemistry; Mary Altz-Smith, MD:
Brookwood Medical Center, Birmingham.
Address reprint requests to Sarah L. Morgan, MD, RD,
Department of Nutrition Sciences, University Station, UAB, Birmingham, AL 35294.
Submitted for publication January 19, 1987; accepted in
revised form May 14, 1987.
Arthritis and Rheumatism, Vol. 30, No. 12 (December 1987)
enzymes required for the biosynthesis of nucleic acids
(2,3). Other folate-dependent enzymes, such as thymidylate synthetase and aminoimidazole carboxamide
ribotide transformylase, are also directly inhibited by
methotrexate (2,4).
In low doses (7.5-15 mg/week), MTX has been
found to be useful in the treatment of a wide variety of
nonmalignant disorders, including psoriasis, polymyositis, dermatomyositis, Reiter’s disease, Wegener’s
granulomatosis, and sarcoidosis (5-14). MTX is a
useful therapy for rheumatoid arthritis (RA) patients;
both controlled studies and studies not using controls
have supported its efficacy in that disease (15-31).
The mechanism of action of low-dose MTX
therapy in RA remains obscure. Weinblatt and coworkers (28) found that rheumatoid factor levels,
tritiated thymidine incorporation by blood mononuclear cells after stimulation with phytohemagglutinin
or concanavalin A, and responses to streptokinasestreptodornase, Cundidu, and purified protein derivative of tuberculin were not significantly different in
MTX-treated patients. Anderson et a1 (30) have reported a marginal decrease in circulating levels of IgG,
IgM, and IgA, and in the Westergren erythrocyte
sedimentation rates, and no differences in levels of C3,
C4, CH50, and thymidine incorporation after phytohemagglutinin or pokeweed mitogen stimulation. Thus,
these experiments suggest that low-dose MTX therapy
has little effect on cellular and humoral immunity.
The toxic side effects of low-dose MTX therapy
often mimic folate deficiency. Signs and symptoms
such as stomatitis, gastrointestinal intolerance, diarrhea, abnormal results on liver function tests, anemia,
and leukopenia have been reported in more than
one-third of RA patients treated with MTX (28,29,32).
In one series, 90% of the RA patients developed
nausea, diarrhea, oral ulcerations, fever, hypersensitivity pneumonitis, or cytopenias (27). All reactions
became mild and tolerable after the dosage was
adjusted. Attempts to lessen toxicity while maximizing
efficacy in RA patients have involved careful monitoring of toxic symptoms, blood counts, liver functions,
and MTX levels (28,30,33,34). With the exception of
one study that found lower lymphocyte folate pools in
psoriasis patients treated with long-term, low-dose
MTX, the size of folate pools has generally not been
moinitored in patients receiving this therapy (35). The
potential significance of monitoring folate levels in RA
is underscored by the fact that at least 2 antifolics,
MTX and sulfasalazine, are effective in the treatment
of IL4 (36-41). This common application suggests that
the inhibition of a common folate-dependent pathway
may be involved in the mechanism of action.
The direct measurement of folate levels in patierits taking MTX is confounded by the fact that it
inhibits the growth of Lactobacillus casei, which is
used in the microbiologic assay of this vitamin (3).
However, folate status can be indirectly evaluated by
an assay of the activity of an enzyme system which
synthesizes serine from glycine and formate, and
requires reduced folate coenzymes (Figure 1). This
activity is called the C, index. We report the activity of
this folate-dependent enzyme system in lymphocytes
of IIA patients treated with low-dose MTX.
Patients. Twenty-nine control subjects and 36 RA
patients were studied at The University of Alabama at
Birmingham Hospitals (UAB) from July 1985 through October 1986. Characteristics of the research subjects are summarized in Table 1. The control subjects were volunteers
frorn the Department of Nutrition Sciences (UAB). They
were free of medical diseases, with the exception of dietcontrolled hypertension in 2 individuals. The RA patients
were drawn from the Rheumatology Clinic. As judged by
their attending rheumatologist, all of the RA patients fulfilled
American Rheumatism Association criteria for definite or
classic rheumatoid arthritis (42).
Sixteen patients were taking MTX (RA + MTX
group). Twenty patients were being treated with other
agents, including gold, penicillamine, aspirin, nonsteroidal
antiinflammatory drugs (NSAID), and prednisone (RA MTX group). The RA + MTX patients were also being
treated with aspirin, NSAID, and prednisone. The mean
cumulative MTX dose in the RA MTX group was 792 mg
( 8-2,073). There was no difference in aspirin, NSAID,
or lprednisone use between groups. There was also no
Figure 1. Folate-dependent pathways involved in the conversion of
glycine and formate to serine. The numbers in circles refer to steps
catalyzed by: 1) dihydrofolate reductase; 2) 10-formyltetrahydrofolate synthetase; 3) 5,lO-methenyltetrahydrofolatecyclohydrolase;
4) 5,lO-methylenetetrahydrofolatedehydrogenase; and 5) serine
hydroxymethyltransferase. Methotrexate (MTX) is shown inhibiting
dihydrofolate reductase, which is required for the conversion of
folic acid (F) or dihydrofolic acid (DHF) to tetrahydrofolic acid
difference in serology, folate vitamin use, or functional class
(as determined by the criteria of Steinbrocker et al [43])
between groups. While dosing schedules were variable, the
blood for assay was generally drawn prior to the next weekly
MTX dose.
Patients were not enrolled in the study if they had not
taken MTX for at least the 2 weeks prior to the blood
drawing. Two of the RA patients had chronic obstructive
pulmonary disease, 2 had diabetes mellitus, 2 had atherosclerotic vascular disease, and 2 had hypertension; all were
medically stable. There were no differences between the
patient groups with regard to chronic diseases. Ten of the 16
RA + MTX patients had toxic side effects with 1 or more of
the following symptoms: stomatitis, nausea, elevated values
on liver function tests, and cytopenias. Three patients were
excluded from the RA + MTX group and from further
analysis because of biochemical or historical evidence of
noncompliance with MTX therapy. Examples of folate levels
in compliant and noncompliant patients are shown in Table 2.
Study design. The study was approved by the Institutional Review Board at UAB. All subjects signed informed
consent forms prior to entry into the study. All subjects were
seen by the same physiciaddietitian. Demographic data
(age, height, weight, medical diagnoses, medications and
Table 1. Characteristics of the control subjects and the rheumatoid arthritis (RA) patients, by group*
Control subjects
(n = 29)
Mean age (range)
Use of vitamins containing folates
(mean 400 pg folate/day)
Mean disease duration, years (range)
No. rheumatoid factor-positive
No. in functional class
33 (21-53)
(n = 16)
(n = 20)
55 (36-70)"
54 (32-74)t
14 (4-35)s
9 (1-18)
792 (8-2,073)
No. who took aspirin andor NSAID
No. who took prednisone
Mean cumulative MTX dose, mg
* RA + MTX (methotrexate) = patients treated with MTX; RA - MTX = patients treated with drugs
other than MTX; NSAID = nonsteroidal antiinflammatory drugs.
t P = 0.03 versus controls, by analysis of variance.
t. P = 0.002 versus controls and RA + MTX group, by chi-square analysis.
§ P = 0.03 versus RA - MTX group, by analysis of variance.
dosages, serology, and functional class) were recorded from
chart review and from patient history. A 24-hour diet recall
was taken, using food models to estimate portion sizes. All
dietary intake data were coded by a method described
previously (44) and entered into an IBM-AT computer.
Assays. Fifty milliliters of venous blood was taken by
sterile technique. Ten milliliters was analyzed for serum
folate, red blood cell (RBC) folate, vitamin B,,, carotene,
vitamin A, vitamin C, thiamine, riboflavin, and vitamin B,
(45-49). This series of assays is referred to as a vitamin
screen. Forty milliliters of blood was required for isolation of
lymphocytes and for assessment of the activity of a series of
folate-requiring enzymes which forms 3(14C)-serine from
14C-formatein the presence of excess glycine (Figure 1). The
activity of the enzyme system, expressed as radioactivity
incorporated into s e i n e (in disintegrations per minute/106
viable lymphocytes) is designated the C, index (35,49-52).
Lymphocytes were isolated by centrifugation in a histopaque gradient (Sigma, St. Louis, MO) (53), and viability
was assayed by trypan blue exclusion (always >95%).
Lymphocytes (1.5-7.0 x 10,) were incubated for 3 hours in
Krebs- Ringer bicarbonate buffer containing excess glycine
and 5 pCi of ''C-formate (Amersham, Arlington Heights,
IL), as described previously (50,511.
The incubations were terminated by acidification,
and the product, I4C-serine, was separated by ion-exchange
chromatography (52). The radioactive p carbon of serine was
liberated, trapped as a formaldehyde-dimedone derivative,
and extracted into toluene, as described previously (503 I ) .
Routinely, the assay was done in triplicate. Duplicate blanks
were terminated immediately after adding radioactivity and
subtracted from assay values. C, indices of normal controls
were compared with lymphocyte, serum, and RBC folate
levels measured by the standard microbiologic method (45).
Lymphocyte homogenates were treated with excess conjugase prior to the microbiologic assay.
Additional experiments were performed to determine
if in vitro extracellular folic acid, folinic acid, or MTX
influenced the C, index. Approximately 2.5 x lo6 viable
lymphocytes were preincubated for 1 hour with increasing
concentrations of folk acid (0-300 ng/ml), folinic acid (0-300
ng/ml), or MTX (0-2.0 mg/ml), followed by the addition of
l4C-formate. The effect of cell number on the C, index was
also tested.
Table 2. Examples of microbiologic assay of folate levels in 2 compliant and 1 noncompliant methotrexate (MTXttreated patients*
Serum folate dilutions (nghl)
Patient 1
Patient 2
Noncompliant patient
RBC folate dilutions (ng/ml)
I :800
* Dilutions refer to the ratio of serum or red blood cell (RBC) folate to Lactobacillus casei growth media. In the absence of MTX (or other L
casei-sensitive antibiotic), folate content is normal and is independent of dilution. The presence of MTX is indicated by increases in apparent
folate content with increasing dilution or by total inhibition.
Statistical analysis. Analysis of variance and least
signiificant difference tests (54) were done on demographic,
vitamin screen, diet intake, and C, index data. Regression
equations and correlation coefficients were calculated for
comparisons of C, index versus lymphocyte folate, RBC
folate, serum folate, and cumulative MTX dose. The significance of the coefficients was determined using a method
described by Pollard (55). When appropriate, the chi-square
test was used on demographic data.
C, index data. It was determined that if at least
1.0 x lo6 cells were used per assay, the C, index
wouild be proportional to the cell number (Figure 2).
Figure 3 shows a plot of C, indices versus pg of
folate/106 viable lymphocytes in control subjects. The
mea.n folate content in lymphocytes in these controls
was 337 pg/106 cells (range 25-850). This value is
higher than the 50 pg reported by Vilter et al (56) and
the 60-123-pg range reported by Colman and Herbert
(57), but is lower than the 547 pg reported by Sullivan
et al (58). In a linear model, in normal subjects, r was
0.70 for C, index versus pg of folate/106 viable lymphocytes, and was statistically significant (P < 0.05).
Plots of C, index versus serum and RBC folate levels
yiellded very weak correlation coefficients of - 0.02
and - 0.25, respectively, which were not statistically
x '01
Figure 2. The effect of cell number on the C, index (dpm/106cells).
The C, index is plotted versus the number of cells per assay. All
cells were obtained from the same individual, and each data point
represents the mean of at least 2 assays.
FOLATE (pg/lO'cells)
Figure 3. Correlation of the C, index (dpm/106 cells) with lymphocyte folate (pg/106 cells) in control subjects. Each data point
represents the mean of triplicate assays of the C, index and
duplicate Lactobacillus casei assays for folate. Linear correlation is
indicated by the solid line (r = 0.70).
significant. Standard deviations of the C, index were
calculated from 3 or more replicates on the same day
and were generally <25% of the mean value. The
day-to-day variations of mean C, indices in the same
individual were within 10%.
Figure 4 shows that the C, index increased in
controls when additional folinic acid (5-formyl-tetrahydrofolic acid; a reduced folate coenzyme) was
added to the system (Figure 4A), but did not increase
when additional folic acid was added to the system
(Figure 4B). Also, in the incubation media, MTX had
no effect on the C, index (Figure 4C).
The C, indices of the control, RA + MTX, and
RA - MTX groups are shown in Figure 5. Even
though the data appeared to be asymmetric, analysis
of variance indicated that the mean C, index was
significantly lower in the RA + MTX group (mean 5
SEM 306 & 5 9 , with a value about one-half that of the
controls (650 80) or of the RA - MTX group (593 5
119) (I' < 0.01). The C, index was weakly correlated
with the cumulative MTX dose, with an r value of
0.34, which was not statistically significant.
0 160
$ 100
X 1000
FOLlNlC ACID ng/ml
F O L I C ACID ng/ml
I .o
2 .o
MTX mg/ml
Figure 4. The effect of folinic acid (5-formyl-tetrahydrofolicacid) (A),
folic acid (B), and methotrexate (MTX) ( C ) on the C, index in normal
control subjects. Folinic acid, folic acid, and MTX were added, at the
concentration shown, to the lymphocyte incubation media 1 hour
prior to addition of I4C-formate.Data are expressed as percentage of
control (no additions during preincubation period). All cells were
obtained from the same individual, and each point represents 1 assay,
Mean serum and red blood cell folate levels were 13.3 ng/ml (range
12.5-14.1) and 352 ng/ml (range 336-372), respectively.
Figure 5. The C, index in 29 control subjects, 20 rheumatoid arthritis
patients treated with drugs other than methotrexate (RA - MTX),
and 16 patients treated with methotrexate (RA + MTX). Means and
standard errors are indicated by solid and dashed lines, respectively.
Each point represents the mean of triplicate assays.
3 groups. While there appear to be large differences in
vitamin B,, and vitamin B, intake between groups,
these differences are not statistically significant because of large standard deviations. Serum vitamin
levels are presented in Table 4. The vitamin screen
values were significantly different between controls
and the 2 RA groups for serum B,, and carotene.
Serum and RBC folate levels could not be determined
in the RA + MTX group by use of the microbiologic
assay, since the growth of L casei is inhibited by MTX
(3). There was no difference between groups for vitamin A, vitamin B,, or vitamin C.
Dietary intake data and vitamin screen data.
Analysis of the 24-hour dietary recalls is presented in
Table 3. Mean nutrient intakes did not differ among the
This report describes the assessment of folate
status in patients with RA treated with MTX. This
enzyme method, known as the C, index, can replace
Table 3. Mean f SD dietary intakes from 24-hour recalls, expressed as percentages of the recommended dietary allowance of each nutrient*
Cirour, (n)
Controls (24)
RA t MTX(16)
RA - MTX (20)
82 f 20
67 ? 18
73 f 18
150 -c 43
120 f 37
141 f 50
Vitamin C
Vitamin A
77 f 77
104 ? 158
89 f 77
189 f 196
425 2 957
469 f 889
218 t 213
316 -+ 362
370 f 542
148 f 115
126 ? 117
259 f 360
90 f 80
562 f 1,340
421 f 844
* RPL+ MTX = rheumatoid arthritis patients treated with methotrexate; RA - MTX = rheumatoid arthritis patients treated with drugs other
than methotrexate. No statistically significant differences in dietary intake were observed (P> 0.05).
the conventional L casei microbiologic assay, which is
inhibited by MTX. In control subjects, the C, index
correlates well with lymphocyte folate levels measured by the standard L casei assay, thus validating
the method; this suggests that the coenzyme level is
the limiting factor. The C, index did not correlate well
with serum or RBC folate levels in control subjects.
This is not surprising, since the C, index reflects the
longer-lived lymphocyte folate stores, whereas the
serum folate level is a measure of recent dietary intake
only and the RBC has a shorter half-life as compared
with the lymphocyte.
The lack of correlation of the C, index with
cumulative MTX dose was somewhat unexpected. It
woiild be reasonable to assume that long-term use of
this drug would continue to deplete pools of reduced
folate coenzymes in the lymphocytes, and would result in a strongly negative correlation coefficient. Since
this was not the case, it may indicate adaptation to
MTX via gene amplification with increased levels of
dihydrofolate reductase (59) or increased rates of
removal of methotrexate polyglutamates from the cell.
It was also of interest that the correlation of C, index
with 1-day folate intake was not significant. This was
not unexpected since the C, index should reflect
long-term folate status and should not vary with shortterm differences in intake. Even though 10 of 16 RA +
MTX patients had toxic symptoms, none had longterm side effects, which suggests that adequate folate
nutriture may be important in modulating side effects
(Table 3).
The results of the incubations with folic acid
and folinic acid added to the medium are in keeping
with the knowledge that reduced folates (i.e., folinic
acid) are required for the enzymatic conversion of
14C-formateand glycine to serine. Alternatively, folinic acid, but not folic acid, may have been taken up by
the lymphocyte. In either case, the C, index is sensitive to pools of reduced folate coenzymes. This underscores the fact that the C, index is an indirect measure
of functionally active intracellular reduced tetrahydrofolate coenzymes having 1-carbon moieties suitable
for biosynthesis of serine in a tissue important in RA.
In contrast, other methods used to assess folate status,
such as radioactive binding assays, urinary formiminoglutamic acid excretion following oral histidine
load, and chromatographic methods, either measure
all forms of folate (both oxidized and reduced), fail to
take into account the levels of folate-dependent enzymes, or lack the sensitivity to be performed in
tissues important in RA, such as lymphocytes or
synovial cells (33).
The C, index is not affected by MTX added to
the medium. This is consistent with observations of
Ellegaard et a1 ( 3 9 , who found that MTX in a concentration of 100 p M to 10 mM had no effect on the C,
index in polymorphonuclear leukocytes. Thus, MTX
is either not taken up by the cells during the preincuba-
Tablle 4. Serum vitamin levels in control subjects and rheumatoid arthritis (RA)patients*
(2-10 nglml)
(n = 29)
(n = 16)
(n = 20)
8.8 t 5.1
(140-369 nglml)
B 12
(200-700 pg/ml)
(0.2-1.5 mgldl)
(25-70 pgldl)
(79-233 pgldl)
(1.15-1.80 N C )
354 -+ 133
0.88 f 0.41
57 f 18
162 f 84
1.54 f 0.27
508 f 267t
0.88 f 0.67
50 t 21
118 -c 54t
1.48 f 0.26
11.9 t 6.6
439 f 260
513 f 195t
1.05 t 0.63
* 20
113 f 45t
1.64 t 0.27
* Values are the mean ? SD; values in parentheses are the normal ranges. RBC = red blood cell; A/C = activity coefficient; RA
patients treated with methotrexate; RA - MTX = patients treated with drugs other than methotrexate.
t P < 0.05 versus controls, by analysis of variance.
+ MTX =
tion period, or short-term exposure to MTX does not
appreciably affect existing pools of intracellular reduced folate coenzymes. A number of cell divisions or
MTX polyglutamation may be necessary to show an
effect of MTX on these folate-requiring enzymes. This
suggests that the C1 index is sensitive to longer-term
exposure to MTX.
Analysis of the 24-hour dietary recall data
showed no differences between groups in the percentage of the recommended dietary allowance consumed,
for calories, protein, folate, vitamin BI2, vitamin C,
and vitamin A. While there are limited data on the
dietary intakes of RA patients, other studies have
shown inadequate intakes of many nutrients, including
vitamin C, vitamin E , and Zn (60-62). The present
data may reflect the bias of a 1-day food record to
underestimate nutrients consumed (63-66), or may
reflect a greater interest among RA patients in consuming adequate diets.
The vitamin level data tend to confirm the diet
intake data, since the vitamin levels, on a whole, did
not differ among the groups. Serum carotene and
vitamin BI2 levels were different between the controls
and the RA patients; however, all values were within
the normal range. Relatively low serum carotene levels may reflect the inability (because of pain and loss of
activities of daily living) of RA patients to prepare and
eat fruits and vegetables. Since vitamin supplement
use was not different between groups, it is not likely to
be the cause of such differences.
While the microbiologic assay of folate is generally not useful in patients treated with antifolics, this
study shows that it can be used as a measure of
compliance with the MTX regimen (Table 2). Normal
serum or RBC folate levels that are independent of
serial dilutions are strong evidence for noncompliance
with the therapy or nonabsorption of MTX.
Folate status as evaulated by the C, index was
MTX group compared with the
poorer in the RA
control and RA - MTX groups. Given the similar
folate intakes between groups, this suggests that MTX
therapy is the reason for the difference. This observation is believed to represent one of the first nutritionrelated abnormalities in an immunologically important
cell line in MTX-treated arthritis patients.
In contrast, proliferation of mitogen-stimulated
lymphocytes is not altered by low-dose MTX (28,30),
which suggests that low doses do not limit folatedependent nucleic acid synthesis. Therefore, it is
intriguing to speculate that the mechanism of action of
low-dose MTX in RA is more closely related to
impaired folate-dependent amino acid metabolism
than it is to nucleic acid metabolism (Figure 1). It may
be that there is a dose-related inhibition of amino acid
and protein metabolism that is below the threshold
needed to inhibit nucleic acid biosynthesis. Evidence
for this hypothesis is given by the fact that sulfasalazine inhibits the folate-dependent conversion of glycine to serine by lymphocytes in vitro (40), and it is
also a useful therapy in RA (36-41).
Finally, it is intriguing to postulate that some
depletion of folate coenzyme levels in lymphocytes
may be necessary for a clinical response in RA,
whereas excessive depletion causes toxic side effects.
The availability of an assay to measure folate status in
these patients may assist in the development of protocols which preserve efficacy of MTX therapy yet
minimize toxicity.
The authors are grateful to the following colleagues
for helpful support and advice during the course of the study:
Drs. W. Koopman, G. Alarch, T. Oglesby, D. Spalding, J.
Reveille, F. O’Sullivan, C. Krumdieck, C. E. Butterworth,
Jr., and R. Weinsier. The expert assistance of Judy Barker,
RN, Ann Snider, LPN, Teresa Dixon, RN, Elizabeth Rodgers, NA, Mary Tittle, Irene Tracey, Harry Vaughn, Anita
Hagood, Connie Robinson, and Renee Redman is also
gratefully acknowledged.
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statue, receiving, terms, patients, dose, low, long, arthritis, methotrexate, therapy, rheumatoid, folate
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