Serum Vitamin C Concentration Is Low in Peripheral Arterial Disease and Is Associated With Inflammation and Severity of Atherosclerosis

 
NEXT SLIDES
Serum Vitamin C Concentration Is Low in Peripheral Arterial Disease and Is Associated With Inflammation and Severity of Atherosclerosis
Serum Vitamin C Concentration Is Low in Peripheral
  Arterial Disease and Is Associated With Inflammation and
                  Severity of Atherosclerosis
               Michel Langlois, MD, PhD; Daniel Duprez, MD, PhD; Joris Delanghe, MD, PhD;
                            Marc De Buyzere, PhD; Denis L. Clement, MD, PhD

Background—Peripheral arterial disease (PAD) is a severe atherosclerotic condition frequently accompanied by
  inflammation and oxidative stress. We hypothesized that vitamin C antioxidant levels might be low in PAD and are
  related to inflammation and disease severity.
Methods and Results—We investigated vitamin C (L-ascorbic acid) levels in 85 PAD patients, 106 hypertensives without
  PAD, and 113 healthy subjects. Serum L-ascorbic acid concentrations were low among PAD patients (median, 27.8
  ␮mol/L) despite comparable smoking status and dietary intake with the other groups (P⬍0.0001). Subclinical vitamin
  C deficiency (⬍11.4 ␮mol/L), confirmed by low serum alkaline phosphatase activity, was found in 14% of the PAD
  patients but not in the other groups. Serum C-reactive protein (CRP) concentrations were significantly higher in PAD
  patients (P⬍0.0001) and negatively correlated with L-ascorbic acid levels (r⫽⫺0.742, P⬍0.0001). In stepwise
  multivariate analysis, low L-ascorbic acid concentration in PAD patients was associated with high CRP level
  (P⫽0.0001), smoking (P⫽0.0009), and shorter absolute claudication distance on a standardized graded treadmill test
  (P⫽0.029).
Conclusions—Vitamin C concentrations are lower in intermittent claudicant patients in association with higher CRP levels
  and severity of PAD. Future studies attempting to relate vitamin C levels to disease occurrence should include in their
  analysis an inflammatory marker such as CRP. (Circulation. 2001;103:1863-1868.)
                      Key Words: arteries 䡲 atherosclerosis 䡲 antioxidants 䡲 inflammation 䡲 claudication

P    eripheral arterial disease (PAD) is a severe atheroscle-
     rotic condition causing intermittent claudication and is
associated with increased cardiovascular and cerebrovascular
                                                                             In this study, we assessed whether inflammation is associ-
                                                                          ated with antioxidant status in patients with intermittent
                                                                          claudication. Vitamin C (L-ascorbic acid) is a water-soluble
morbidity and mortality.1,2 Moreover, it has a major impact               antioxidant capable of scavenging free radicals and is the
on the quality of life because of the reduction in walking                first-line defense in the control of the redox state, sparing
ability. In clinical practice, PAD is assessed noninvasively by           other endogenous antioxidants from consumption.10,11 We
the determination of the ankle/brachial systolic blood pres-              tested the hypothesis that vitamin C status might be low in
sure index (ABI) and absolute claudication distance (ACD)                 PAD and that it correlates with measures of inflammation and
on a treadmill test, a parameter of functional adaptation to the          functional status in patients with intermittent claudication.
disease.3–5
   Smoking, diabetes mellitus, hyperlipidemia, and hyperten-                                          Methods
sion are the key risk factors for PAD,6 but it is now equally
recognized that low-grade inflammation contributes impor-                 Study Groups
                                                                          We investigated 85 PAD patients (Fontaine stage II) who were
tantly to the initiation and the progression of the vascular              regularly followed for at least 12 months at our department, 106
atherosclerotic lesions. Specifically, slightly elevated levels           patients with essential hypertension without PAD (to account for the
of C-reactive protein (CRP), an acute phase reactant, are                 effect of pulse pressure), and 113 healthy volunteers of the same
associated with PAD and are found among apparently healthy                region. Hypertensives were defined along the WHO/ISH criteria for
subjects at risk for developing future PAD.7,8 It has been                at least 12 months and were receiving treatment with antihyperten-
                                                                          sive drugs.12 Patients with either surgical revascularization or per-
suggested that inflammation within the atherosclerotic lesions
                                                                          cutaneous transluminal angioplasty procedures at the lower limb
and the subsequent release of free radicals by phagocytes                 arteries were excluded, as well as patients with acute myocardial
result in oxidative stress that further enhances vascular                 infarction, angina pectoris, heart failure, coronary revascularization
damage.9                                                                  procedures, or cerebrovascular events during the last 6 months. Other

  Received September 28, 2000; revision received December 21, 2000; accepted December 21, 2000.
  From the Departments of Clinical Chemistry (M.L., J.D.) and Cardiology (D.D., M.D.B., D.L.C.), Ghent University Hospital, Ghent, Belgium.
  Correspondence to Dr Michel Langlois, Laboratory of Clinical Chemistry, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
E-mail michel.langlois@rug.ac.be
  © 2001 American Heart Association, Inc.
  Circulation is available at http://www.circulationaha.org

                                                                   1863
                               Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
1864       Circulation            April 10, 2001

                            TABLE 1.       Clinical Characteristics of Study Groups
                                                               Healthy Subjects      Hypertension Without PAD
                                                                  (n⫽113)                    (n⫽106)                 PAD (n⫽85)
                            Age, y                                  61⫾12                      62⫾14                    68⫾10
                            Male sex, %                                45                         42                      78†
                            Postmenopausal, % of women                 89                         87                      89
                            Body mass index, kg/m2                  25⫾4                       28⫾5                     26⫾5
                            Hypertension, %                            0                         100                      50
                            Diabetes mellitus, %                       0                          19                      17
                            Smoking, %                                 31                         34                      33
                            No. of cigarettes/d                     22⫾9                       19⫾8                     18⫾8
                            Vitamin C intake, mg/d                 121⫾39                     117⫾36                   118⫾40
                            Physical activity, MET h/wk*            27⫾16                      24⫾15                    20⫾13‡
                            Blood pressures, mm Hg
                              Systolic                             126⫾10                     147⫾15                   142⫾20‡
                              Diastolic                             75⫾8                       91⫾12                    82⫾9‡
                              Pulse pressure                        51⫾9                       56⫾13                    60⫾19‡
                            ABI                                       ⬎1                         ⬎1                   0.61⫾0.15
                            Medication, % of patients
                              ␤-Blockers                               0                          64                      31
                              Calcium antagonists                      0                          44                      21
                              ACE inhibitors                           0                          35                      19
                              Diuretics                                0                          29                      11
                              Aspirin                                  0                          0                       68
                              Lipid-lowering agents                    0                          20                      19
                              Values are mean⫾SD or percentages.
                              *Intensity (MET, metabolic units) multiplied by the duration (h/wk) of specified activities.16
                              †P⬍0.001 (␹2 test).
                              ‡P⬍0.05 (Kruskal-Wallis test).

exclusion criteria were intake of antioxidant vitamin supplements,                 tration was calculated by the Friedewald formula. Diabetes control
alcohol intake ⬎20 g/d, and hormone replacement therapy. PAD                       status was assessed by hemoglobin A1c (HbA1c) with a Variant II
patients had been motivated to participate in an exercise rehabilita-              chromatographic system (Bio-Rad). Intra-assay coefficients of vari-
tion program that included 3 walking sessions per week with a                      ation were ⬍5.0% for L-ascorbic acid, ⬍1.3% for alkaline phospha-
duration ⬎30 minutes per session, with near maximal pain during                    tase, ⬍3.8% for CRP, ⬍4.4% for fibrinogen, ⬍1.8% for lipids, and
training as the end point and program length ⬎6 months.13,14                       ⬍1.5% for HbA1c.
   Table 1 summarizes clinical characteristics of the study groups. A
dietary history was taken to estimate the amount of vitamin C                      Vascular Investigations
ingested by means of a food frequency questionnaire according to
                                                                                   After blood sampling, all subjects underwent clinical examinations
Donnan et al.15 Intensity and duration of physical activity were also
                                                                                   including blood pressure measurements at the left and right brachial
assessed with a questionnaire.16 Smoking status was classified as
current smokers and nonsmokers; individuals who stopped smoking                    artery by sphygmomanometry (3 times in sitting position with
within 4 weeks before enrollment in the study were excluded to                     2-minute intervals) and 12-lead ECG. In PAD patients, arterial
account for the effect of smoking cessation on vitamin C status.17                 systolic blood pressures at the left and the right brachial and posterior
Informed consent was obtained from all subjects, and the study was                 tibial arteries were measured by a Doppler 8-MHz ultrasound device
approved by the ethics committee of Ghent University Hospital.                     (Scimed Digitop 840S). ABI was calculated for both legs, and the
                                                                                   lowest ABI was taken as the study parameter.
Biochemical Investigations                                                            Thereafter, PAD patients (n⫽70) who did not have contraindica-
Fasting blood samples were taken between 8:00 and 9:00 AM. None                    tions to performing a walking test (chronic obstructive pulmonary
of the subjects took any medication during the last 12 hours before                disease, cardiac rhythm disturbances, orthopedic or neurological
sampling. Within 30 minutes, serum vitamin C (L-ascorbic acid)                     conditions, ulcera) underwent a standardized graded treadmill pro-
concentrations were measured by the ascorbate oxidase method on a                  tocol with a speed of 3 km/h and adjustable grade. For the first 5
Hitachi 911 analyzer (Roche).18 Serum alkaline phosphatase activity,               minutes, the patients had to walk at a grade of 0%, then the grade
a biochemical marker of vitamin C deficiency,19 was measured with                  increased every 5 minutes by 5% up to 15%.20,21 All patients were
commercial reagents on a Hitachi 747 analyzer (Roche). Serum CRP                   accustomed to treadmill tests and had at least 3 tests during the last
concentrations were measured by high-sensitive latex-enhanced                      12 months. ACD on the present treadmill test was taken as study
immunonephelometry (BN II nephelometer, Dade Behring). Plasma                      parameter. Clinical characteristics of patients who participated in the
fibrinogen was assayed by prothrombin time– derived measurements                   treadmill protocol were age, 69⫾9 years; male sex, 77%; body mass
on an ACL-200 analyzer (Instrumentation Laboratory). Serum total                   index, 26⫾4 kg/m2; smokers, 36%; diabetes mellitus, 11%; hyper-
cholesterol, HDL cholesterol, and triglycerides were assayed with                  tension, 53%; pulse pressure, 59⫾19 mm Hg; ABI, 0.60⫾0.14; and
commercial reagents (Hitachi 747, Roche); LDL cholesterol concen-                  ACD, 389⫾165 m.

                               Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
Langlois et al             Low Vitamin C Concentration in PAD             1865

                    TABLE 2.       Vitamin C Levels, Inflammatory Markers, Lipids, and Diabetes Control
                                                Healthy Subjects   Hypertension Without                         Wilcoxon Test*
                                                   (n⫽113)            PAD (n⫽106)              PAD (n⫽85)             (P )
                    L-Ascorbic   acid, ␮mol/L   51.7 (42.8–63.5)       49.6 (38.1–62.5)      27.8 (15.8–42.5)     ⬍0.0001
                    CRP, mg/L                   2.51 (0.78–4.16)       2.32 (1.28–3.58)      4.80 (2.00–9.55)     ⬍0.0001
                    Fibrinogen, g/L             3.12 (2.35–3.86)       3.64 (2.99–4.84)      4.95 (3.99–5.73)       0.002
                    Cholesterol, mmol/L         5.21 (4.58–6.20)       5.22 (5.15–6.55)      5.78 (5.19–6.87)       0.003
                    HDL cholesterol, mmol/L     1.52 (1.21–1.82)       1.32 (1.15–1.62)      1.04 (0.79–1.30)       0.0004
                    LDL cholesterol, mmol/L     3.47 (2.51–4.04)       3.60 (2.98–4.22)      4.30 (3.60–5.05)       0.0003
                    Triglycerides, mmol/L       1.11 (0.88–1.71)       1.48 (1.05–1.92)      2.20 (1.27–2.86)       0.0003
                    HbA1c,† %                           䡠䡠䡠             7.6 (6.4–9.3)         7.4 (7.0–8.8)            NS
                      Data are median (interquartile range).
                      *Kruskal-Wallis test: P⬍0.05 for L-ascorbic acid, inflammatory parameters, and lipids. Subtesting PAD vs
                    hypertension without PAD (Wilcoxon test).
                      †Hemoglobin A1c in diabetic patients (20 hypertensives without PAD, 14 PAD patients).

Statistics                                                                    Inflammatory Markers
Values are expressed as mean⫾SD or median and interquartile                   Biochemical markers of inflammation (CRP, fibrinogen)
ranges. Statistical differences were evaluated with the Wilcoxon              were higher in PAD patients than in the other study groups
test for comparison between 2 groups, the Kruskal-Wallis test for
                                                                              (P⬍0.005, Table 2). In the PAD group, serum CRP correlated
comparison between ⬎2 groups, or the ␹2 test. PAD patients were
stratified for L-ascorbic acid levels with the lower reference limit          with plasma fibrinogen (r⫽0.600, P⬍0.0001); no significant
(28.4 ␮mol/L) and the limit for vitamin C deficiency (11.4                    associations were observed with age, sex, body mass index,
␮mol/L).22 The median CRP and fibrinogen levels were used to                  physical activity, HbA1c, blood pressures, or smoking status
categorize PAD patients above and below these levels. Correla-                of the PAD patients. Serum CRP and plasma fibrinogen were
tions between data were examined by Spearman rank correlation
                                                                              less elevated in PAD patients taking aspirin (Table 3).
coefficients. Stepwise multivariate analysis was performed with
L-ascorbic acid as the dependent variable. Statistical analysis was
                                                                                 Serum L-ascorbic acid concentration in PAD patients
carried out with MEDCALC and SPSS 9.0 software. Probability                   showed a negative correlation with serum CRP (r⫽⫺0.742,
values ⬍0.05 were considered statistically significant.                       P⬍0.0001) (Figure 2) and plasma fibrinogen (r⫽⫺0.387,
                                                                              P⬍0.0001) levels. After adjustment for smoking and aspirin
                             Results                                          intake, a relative risk for vitamin C deficiency of 1.68 (95%
Vitamin C Levels                                                              CI, 1.27 to 2.21) was calculated among PAD patients with
Table 2 summarizes biochemical data of the study groups.                      serum CRP concentration ⬎4.80 mg/L (median value). Se-
Serum L-ascorbic acid concentrations were comparable                          rum L-ascorbic acid levels in the PAD subgroup with CRP
among healthy subjects and hypertensives without PAD but                      concentration ⱕ4.80 mg/L were comparable to those in
were much lower in the PAD group (P⬍0.0001): 52% of the                       hypertensives without PAD and healthy subjects.
PAD patients had L-ascorbic acid levels below the lower
reference limit (28.4 ␮mol/L), and 14% showed biochemical
evidence of vitamin C deficiency (⬍11.4 ␮mol/L). In the
latter subgroup of patients, serum alkaline phosphatase activ-
ity was very low compared with PAD patients with L-ascorbic
acid levels ⱖ11.4 ␮mol/L (Figure 1): 21.3 (9.1 to 45.4) U/L
versus 64.8 (50.0 to 76.7) U/L, respectively (P⬍0.0001).
   Smoking was comparable between the study groups and
was associated with ⬇40% lower serum L-ascorbic acid
concentrations in PAD patients (P⫽0.0005, Table 3), hyper-
tensives (P⫽0.0013), and healthy subjects (P⫽0.0002). No
significant associations of vitamin C levels with age, sex,
body mass index, physical activity, HbA1c, or blood pres-
sures were observed in the study groups. Serum L-ascorbic
acid concentration correlated with dietary vitamin C intake
only in healthy subjects (r⫽0.297, P⫽0.004) and hyperten-
sives without PAD (r⫽0.261, P⫽0.006). PAD patients taking                     Figure 1. Box-and-whisker plots of serum alkaline phosphatase
aspirin showed a less pronounced reduction of serum                           activity in PAD patients (n⫽85) according to serum L-ascorbic
L-ascorbic acid levels (Table 3); vitamin C levels were not                   acid concentration, categorized by lower reference limit (28.4
                                                                              ␮mol/L) and limit for subclinical vitamin C deficiency (11.4
different in PAD patients taking antihypertensive drugs than                  ␮mol/L). Boxes show median and quartiles; whiskers are 5th
in those without antihypertensive treatment.                                  and 95th percentiles.

                                 Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
1866      Circulation           April 10, 2001

             TABLE 3.       Effects of Smoking and Aspirin Intake in PAD Patients
                                                            Smoking                                          Aspirin Intake†

                                           No (n⫽57)           Yes (n⫽28)         P*        No (n⫽27)              Yes (n⫽58)        P*
             L-Ascorbic                37.5 (16.2–52.2)     22.2 (14.8–26.7)    0.0005   18.7 (13.9–29.0)       35.8 (17.2–47.1)    0.0004
             acid, ␮mol/L
             CRP, mg/L                   4.78 (2.00–9.39)    5.34 (2.05–9.85)     NS      8.78 (3.98–11.5)       3.24 (1.66–6.65)   0.0014
             Fibrinogen, g/L             4.86 (3.90–5.73)    5.13 (4.25–5.74)     NS      5.07 (4.11–6.03)       4.70 (3.75–5.54)   0.032
               Data are median (interquartile range).
               *Wilcoxon test.
               †160 mg/d.

Serum Lipids                                                                    ence limit) were characterized by a lower ABI (P⫽0.0007)
Serum concentrations of total cholesterol, LDL cholesterol,                     and a shorter ACD (P⫽0.0001). These differences were
and triglycerides were higher (P⬍0.005) among PAD pa-                           significant in both smoking and nonsmoking patients. Ele-
tients, whereas HDL cholesterol levels were lower                               vated inflammatory markers were also associated with low
(P⬍0.0005) compared with the other groups (Table 2). In the                     ABI and ACD; no effects of serum lipids were observed. ABI
PAD group, serum HDL cholesterol positively correlated                          correlated with serum L-ascorbic acid (r⫽0.406, P⫽0.001),
with serum L-ascorbic acid (r⫽0.362, P⬍0.0001) and nega-                        serum CRP (r⫽⫺0.429, P⬍0.0001), and plasma fibrinogen
tively with serum CRP (r⫽⫺0.437, P⫽0.003). Serum                                (r⫽⫺0.397, P⫽0.009). Similarly, ACD correlated with se-
L-ascorbic acid concentration did not correlate with other                      rum L-ascorbic acid (r⫽0.552, P⬍0.0001), serum CRP
lipids (total and LDL cholesterol, triglycerides).                              (r⫽⫺0.476, P⬍0.0001), and plasma fibrinogen (r⫽⫺0.426,
                                                                                P⫽0.006).
L-Ascorbic    Acid Stability                                                       Pulse pressure of PAD patients showed a weak negative
In serum from male nonsmokers (15 PAD patients, 10 healthy                      correlation with serum L-ascorbic acid (r⫽⫺0.247, P⫽0.048).
control subjects), vitamin C (Fluka) was added to increase its                  In stepwise multivariate analysis (r⫽0.798, P⫽0.0001 for the
concentration by 114 ␮mol/L (20 mg/L). The sera were then                       model), only CRP, smoking, and ACD were significantly related
incubated at 37°C for 4 hours and L-ascorbic acid concentra-                    to vitamin C levels in PAD (Table 5).
tions were measured (in triplicate) at baseline and at the end
of the experiment. Baseline L-ascorbic acid concentrations in                                                Discussion
the spiked sera were 127.5⫾5.8 ␮mol/L (PAD) and                                 We found that circulating vitamin C (L-ascorbic acid) con-
132.4⫾6.2 ␮mol/L (control subjects). After 4 hours, the                         centrations are depleted in PAD patients, whereas levels of
decrease of L-ascorbic acid concentration was significantly                     the antioxidant are comparable between hypertensives with-
higher in sera from PAD patients (57⫾13%) than in control                       out PAD and healthy subjects. Vitamin C is the most effective
sera (40⫾8%) (P⫽0.02). The L-ascorbic acid decrease was                         defense against free radicals in the blood and is the first
even more striking (68%) in serum from PAD patients with                        antioxidant to be used up during oxidative stress, thereby
high CRP concentration (range, 8.1 to 12.3 mg/L), whereas                       sparing other endogenous antioxidants.10,11 Therefore, our
depletion of the vitamin in patient sera with lower CRP level                   finding suggests a higher oxidative stress in patients with
(range, 1.5 to 4.2 mg/L) was not significantly different than in
control sera (Figure 3).

Vascular Investigations
Table 4 illustrates the effect of vitamin C levels and inflam-
mation on ABI and ACD. PAD patients with serum
L-ascorbic acid concentrations ⬍28.4 ␮mol/L (lower refer-

                                                                                Figure 3. L-ascorbic acid depletion in spiked sera from non-
                                                                                smoking men (10 control subjects, 15 PAD patients), stratified
                                                                                for serum CRP concentration above and below level of 5.0
                                                                                mg/L. Shown is percentage decrease of L-ascorbic acid con-
Figure 2. Correlation between serum L-ascorbic acid and CRP                     centration (mean⫾SD) after incubation of sera for 4 hours
concentrations in PAD patients (r⫽⫺0.742, P⬍0.0001).                            at 37°C.

                                Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
Langlois et al      Low Vitamin C Concentration in PAD                  1867

TABLE 4. Effect of Vitamin C Level and Inflammation on                                 acid levels in PAD only correlated with HDL cholesterol, a
Parameters of PAD                                                                      negative acute phase reactant,27 and not with other serum
                                                                     ACD, m            lipids. Other confounders may be the predominance of men in
                                  ABI (n⫽85)                         (n⫽70)            the PAD group and the differences in physical activity and in
  L-Ascorbic   acid, ␮mol/L
                                                                                       prevalence of antihypertensive treatment between groups, but
                                                                                       we found no significant associations of vitamin C status with
     ⬍28.4*                    0.55 (0.46–0.61)     ⬍28.4*       325 (175–400)
                                                                                       sex, physical activity, or intake of antihypertensive drugs.
     ⱖ28.4                     0.65 (0.59–0.73)     ⱖ28.4        488 (375–600)
                                                                                       Potential confounding by nutrient intake15 is limited because
                                  P⫽0.0007                          P⫽0.0001           all patients had a regular dietary intake of fruits and vegeta-
  CRP, mg/L                                                                            bles, and none of the patients received antioxidant
     ⱕ4.80†                    0.64 (0.57–0.73)       ⱕ4.84†     482 (375–588)         supplements.
     ⬎4.80                     0.55 (0.46–0.63)       ⬎4.84      325 (150–400)            In vitro, stability of L-ascorbic acid is low in serum from
                                  P⫽0.0042                          P⫽0.0001           PAD patients with high CRP levels, suggesting a higher
  Fibrinogen, g/L
                                                                                       degree of oxidative stress associated with inflammation. It is
                                                                                       unclear whether vitamin C depletion in atherosclerosis is
     ⱕ4.95†                    0.63 (0.53–0.74)       ⱕ4.89†     425 (325–518)
                                                                                       mainly a cause or a result of the inflammatory disease,
     ⬎4.95                     0.57 (0.48–0.66)       ⬎4.89      350 (150–475)
                                                                                       although both may be true. Vitamin C depletion is athero-
                                   P⫽0.041                          P⫽0.038            genic by increasing the susceptibility of LDL cholesterol to
  Data are median (interquartile range).                                               oxidation and is associated with increased cardiovascular
  *Lower reference limit.                                                              risk,22 but it is now equally recognized that reactive oxygen
  †Median value in the patient group.                                                  species formed by the inflammatory response in an existing
                                                                                       atherosclerotic lesion may in turn reduce vitamin C antioxi-
PAD. Smoking, a key risk factor in PAD and known to reduce                             dant levels.9
vitamin C levels,17 was comparable in the 3 study groups, and                             Vitamin C depletion affects the functional state of the
vitamin C depletion was also observed among nonsmoking                                 peripheral circulation, as evidenced by low ACD. Smoking
PAD patients. Approximately 50% of the PAD patients had                                has been demonstrated to affect walking distance,28 but we
vitamin C levels below the lower reference limit, and ⬎10%                             found that the relation between vitamin C depletion and low
had vitamin C deficiency. Low serum alkaline phosphatase                               ACD was also consistent among nonsmoking PAD patients.
activities, a biochemical sign of subclinical vitamin C defi-                          A number of studies have compared the reproducibility of the
ciency, were found in the latter subgroup of patients.19                               initial and absolute claudication distance, with most demon-
   Vitamin C depletion in PAD is related to low-grade                                  strating that ACD is more reproducible and therefore presum-
inflammation. We found that serum L-ascorbic acid levels                               ably the more appropriate measurement to use as a primary
inversely correlated with serum CRP and plasma fibrinogen                              end point.20,21 ACD also has the theoretical justification that
concentrations in PAD patients, similar to what has been                               it probably more truly represents real life, in which the patient
observed in other clinical settings.23,24 Low vitamin C status                         is likely to walk even after the first appearance of claudication
has been observed in critically ill patients and could not be                          discomfort.
prevented by the use of parenteral nutrition containing ascor-                            In stepwise multivariate analysis, ACD is a determinant of
bic acid.24 Other authors have proposed that low-dose aspirin                          vitamin C concentration in PAD patients, whereas other
treatment might reduce CRP levels25 and oxidative stress.26 In                         vascular parameters (ABI, pulse pressure) failed to enter the
this study, intake of aspirin (160 mg/d) was not independently                         model. We found that low ABI is related to inflammation,
associated with vitamin C concentration in stepwise multi-                             similar to what has been observed in previous studies.29,30
variate analysis.                                                                         The question arises whether antioxidant vitamin supple-
   Because PAD patients show a less favorable lipid profile,                           ments would be useful to address reduction of oxidative stress
depressed vitamin C status may be a reflection of their                                in PAD. A randomized controlled trial has suggested that
hyperlipidemia. However, we found that serum L-ascorbic                                antioxidants may prevent cardiovascular events in PAD
                                                                                       patients but do not improve lower limb function.31 Recent
TABLE 5. Stepwise Multivariate Analysis With L-Ascorbic Acid                           trials showed no benefit of vitamin E in the treatment for
as Dependent Variable*
                                                                                       intermittent claudication.32–34 An ongoing multicenter Euro-
        Variable                       ␤-Coefficient (SE)             P                pean trial, the Critical Leg Ischemia Prevention Study
        Log(CRP), mg/L                 ⫺11.862 (1.589)            0.0001*              (CLIPS), investigates the effectiveness of low-dose aspirin
        Smoking (yes vs no)             ⫺9.828 (2.819)            0.0009*
                                                                                       and antioxidant vitamins (vitamin E, vitamin C, ␤-carotene)
                                                                                       with a 2⫻2 factorial design. However, vitamin C adminis-
        ACD, m                             0.021 (0.009)          0.029
                                                                                       tered as a dietary supplement to humans has been shown to
   This analysis was performed in PAD patients who performed a treadmill test          exhibit pro-oxidant properties, which may give rise to para-
(n⫽70).
   *Also significant in a model for all PAD patients (n⫽85) (ACD not included).
                                                                                       doxical effects in clinical intervention trials.35
Variables that failed to enter the model: alkaline phosphatase, fibrinogen, HDL
cholesterol, dietary vitamin C intake, physical activity, aspirin intake, ABI, pulse   Summary
pressure.                                                                              Vitamin C concentrations are low in PAD and are associated
   r⫽0.798 for the model, F⫽38.4 (P⫽0.0001).                                           with inflammation and the patient’s functional state. Low-

                                     Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
1868         Circulation            April 10, 2001

grade inflammation in atherosclerosis may be associated with                      15. Donnan PT, Thomson M, Fowkes FG, et al. Diet as a risk factor for
oxidative stress and the resultant decrease in antioxidants                           peripheral arterial disease in the general population: the Edinburgh Artery
                                                                                      Study. Am J Clin Nutr. 1993;57:917–921.
such as vitamin C. Future studies attempting to relate circu-                     16. Lakka TA, Venäläinen JM, Rauramaa R, et al. Relation of leisure-time
lating vitamin C concentrations to disease occurrence should                          physical activity and cardiorespiratory fitness to the risk of acute myo-
include in their analysis a marker of inflammatory response                           cardial infarction in men. N Engl J Med. 1994;330:1549 –1554.
                                                                                  17. Lykkesfeldt J, Priemé H, Loft S, et al. Effect of smoking cessation on
such as CRP.                                                                          plasma ascorbic acid concentration. BMJ. 1996;313:91. Letter.
                                                                                  18. Beutler HO. L-ascorbate and L-dehydroascorbate. In: Bergmeier HU, ed.
                        Acknowledgments                                               Methods of Enzymatic Analysis. Vol VI. Weinheim, Germany: Verlag
Michel Langlois is recipient of a postdoctoral fellowship of the Fund                 chemie; 1984:376 –385.
for Scientific Research (FWO), Flanders (Belgium). We are grateful                19. Friedman RB, Young DS, eds. Effects of Disease on Clinical Laboratory
to Dirk De Bacquer, An Maas, Annie Van Wassenhove, and Inge                           Tests. Washington, DC: AACC Press; 1989.
                                                                                  20. Chandry H, Holland A, Dormandy J. Comparison of graded versus
Van Pottelbergh for their advice and skillful assistance.
                                                                                      constant treadmill test protocols for quantifying intermittent claudication.
                                                                                      Vasc Med. 1997;2:93–97.
                              References                                          21. Gardner A, Skinner JS, Cantwell BW, et al. Progressive versus
 1. Criqui MH, Fronek A, Barret-Conner E, et al. The prevalence of periph-            single-stage treadmill tests for evaluation of claudication. Med Sci Sports
    eral arterial disease in a defined population. Circulation. 1985;71:              Exerc. 1991;23:402– 408.
    510 –515.                                                                     22. Nyyssönen K, Parviainen MT, Salonen R, et al. Vitamin C deficiency and
 2. Hiatt WR, Hirsch AT, Regensteiner JG, et al. Clinical trials for claudi-          risk of myocardial infarction: prospective population study of men from
    cation: assessment of exercise performance, functional status, and clinical       eastern Finland. BMJ. 1997;314:634 – 638.
    end points. Circulation. 1995;92:614 – 621.                                   23. Galloway P, McMillan DC, Sattar N. Effect of the inflammatory response
 3. Leng GC, Fowkes FG, Lee AJ, et al. Use of ankle brachial pressure index           on trace element and vitamin status. Ann Clin Biochem. 2000;37:
    to predict cardiovascular events and death: a cohort study. BMJ. 1996;            289 –297.
    313:1440 –1444.                                                               24. Schorah CJ, Downing C, Piripitsi A, et al. Total vitamin C, ascorbic acid,
 4. Newman AB, Shemanski L, Manolio TA, et al. Ankle-arm index as a                   and dehydroascorbic acid concentrations in critically ill patients. Am J
    predictor of cardiovascular disease and mortality in the Cardiovascular           Clin Nutr. 1996;63:760 –765.
    Health Study: the Cardiovascular Health Study Group. Arterioscler             25. Ikonomidis I, Andreotti F, Economou E, et al. Increased proinflammatory
    Thromb Vasc Biol. 1999;19:538 –545.                                               cytokines in patients with chronic stable angina and their reduction by
 5. Duprez D, De Backer T, De Buyzere M, et al. Estimation of walking                 aspirin. Circulation. 1999;100:793–798.
    distance in intermittent claudication: need for standardization. Eur          26. Ristimäe T, Zilmer M, Zilmer K, et al. Effect of low-dose aspirin on the
    Heart J. 1999;20:641– 644.                                                        markers of oxidative stress. Cardiovasc Drugs Ther. 1999;13:485– 490.
 6. Criqui MH, Denenberg JO, Langer RD, et al. The epidemiology of                27. Hallfrisch J, Singh VN, Muller DC, et al. High plasma vitamin C asso-
    peripheral arterial disease: importance of identifying the population at          ciated with high plasma HDL- and HDL2 cholesterol. Am J Clin Nutr.
    risk. Vasc Med. 1997;2:221–226.                                                   1994;60:100 –105.
 7. Erren M, Reinecke H, Junker R, et al. Systemic inflammatory parameters        28. Cahan MA, Montgomery P, Otis RB, et al. The effect of cigarette
    in patients with atherosclerosis of the coronary and peripheral arteries.         smoking on six-minute walk distance in patients with intermittent clau-
    Arterioscler Thromb Vasc Biol. 1999;19:2355–2363.                                 dication. Angiology. 1999;50:537–546.
 8. Ridker PM, Cushman M, Stampfer MJ, et al. Plasma concentrations of            29. Lowe GD, Fowkes FG, Dawes J, et al. Blood viscosity, fibrinogen, and
    C-reactive protein and risk of developing peripheral vascular disease.            activation of coagulation and leukocytes in peripheral arterial disease and
    Circulation. 1998;97:425– 428.                                                    the normal population in the Edinburgh Artery Study. Circulation. 1993;
 9. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;             87:1915–1920.
    340:115–126.                                                                  30. Tracy RP, Psaty BM, Macy E, et al. Lifetime smoking exposure affects
10. Frei B, England L, Ames BN. Ascorbic acid is an outstanding anti-                 the association of C-reactive protein with cardiovascular disease risk
    oxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989;86:                 factors and subclinical disease in healthy elderly subjects. Arterioscler
    6377– 6381.                                                                       Thromb Vasc Biol. 1997;17:2167–2176.
11. Jialal I, Grundy SM. Preservation of the endogenous antioxidants in low       31. Leng GC, Lee AJ, Fowkes FG, et al. Randomized controlled trial of
    density lipoprotein by ascorbate but not probucol during oxidative mod-           antioxidants in intermittent claudication. Vasc Med. 1997;2:279 –285.
    ification. J Clin Invest. 1991;87:597– 601.                                   32. Pryor WA. Vitamin E and heart disease: basic science to clinical inter-
12. 1999 World Health Organization. International Society of Hypertension             vention trials. Free Radic Biol Med. 2000;141–164.
    guidelines for the management of hypertension. J Hypertens. 1999;17:          33. Yusuf S, Dagenais G, Pogue J, et al. Vitamin E supplementation and
    151–183.                                                                          cardiovascular events in high-risk patients: the Heart Outcome Prevention
13. Remijnse-Tamerius HCM, Duprez D, De Buyzere M, et al. Why is                      Evaluation Study Investigators. N Engl J Med. 2000;342:154 –160.
    training effective in the treatment of patients with intermittent claudi-     34. Kleijnen J, Mackerras D. Vitamin E for intermittent claudication
    cation? Int Angiol. 1999;18:103–112.                                              (Cochrane Review). In: The Cochrane Library. Issue 3. Oxford, UK:
14. Regensteiner J, Gardner A, Hiatt W. Exercise testing and exercise reha-           Update Sofware; 1999.
    bilitation for patients with peripheral arterial disease: status 1997. Vasc   35. Podmore ID, Griffiths HR, Herbert KE, et al. Vitamin C exhibits pro-
    Med. 1997;2:147–155.                                                              oxidant properties. Nature. 1998;392:559. Letter.

                                   Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
Serum Vitamin C Concentration Is Low in Peripheral Arterial Disease and Is Associated With
                        Inflammation and Severity of Atherosclerosis
     Michel Langlois, Daniel Duprez, Joris Delanghe, Marc De Buyzere and Denis L. Clement

                                         Circulation. 2001;103:1863-1868
                                         doi: 10.1161/01.CIR.103.14.1863
         Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
                          Copyright © 2001 American Heart Association, Inc. All rights reserved.
                                     Print ISSN: 0009-7322. Online ISSN: 1524-4539

The online version of this article, along with updated information and services, is located on the World
                                              Wide Web at:
                                      http://circ.ahajournals.org/content/103/14/1863

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once
the online version of the published article for which permission is being requested is located, click Request Permissions in
the middle column of the Web page under Services. Further information about this process is available in thePermissions
and Rights Question and Answer document.

Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints

Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/

                          Downloaded from http://circ.ahajournals.org/ by guest on September 27, 2015
You can also read
NEXT SLIDES ... Cancel