Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989), pp. 559-566
Ascorbic Acid Absorption in Crohn's Disease Studies Using L-[carboxyl-14C]AscorbicAcid STEPHEN H. PETTIT, MA, ChM, FRCS, JONATHON L. SHAFFER, MRCP, C. WENDY JOHNS, MA, RAYMOND J.M. BENNETT, BSc, PhD, and MILES H. IRVING, MD, ChM, FRCS
Total body pool and intestinal absorption of ascorbic acid were studied in 12 patients undergoing operation for Crohn's disease (six with fistulae and six without) and in six control patients undergoing operation for reasons other than Crohn's disease. L[carboxyl-14C]Ascorbic acid, 0.19-0.40 megabecquerels (MBq), was given orally. After a period o f equilibration, the labeled ascorbic acid was flushed out o f the patient's body tissues using large doses of unlabeled ascorbic acid. Intestinal absorption o f ascorbic acid, assessed from the total cumulative urinary 14Crecovery, was found to be similar in patients with fistulizing Crohn's disease (73.9 +- 8.45%), those without fistulas (72.8 + 11.53%), and in controls (80.3 +- 8.11%). Total body pools o f ascorbic acid, calculated using the plasma 14C decay curves, were similar in patients with Crohn's disease with fistulas (17.1 +- 5.91 mg/kg), patients without fistulas (9.6 +- 3.58 mg/kg), and in controls (13.3 +- 4.28 mg/kg). The results indicate that ascorbic acid absorption is normal in patients with both fistulizing and nonfistulizing Crohn's disease. The results suggest that routine supplements o f vitamin C are not necessary unless oral ascorbic acid intake is low. K E Y WORDS: Crohn's disease; [carboxyl-14C]ascorbic acid.
ascorbic acid absorption; total body pool of ascorbic acid; L-
There have been several reports indicating that patients with Crohn's disease have lower plasma and leukocyte ascorbic acid levels than healthy controls (1-4). Linaker (4) suggested that ascorbic acid absorption was impaired in patients with Crohn's disease as the dietary intake of ascorbic acid was similar in patients and controls. This has Manuscript received September 23, 1987; revised manuscript received March 28, 1988; accepted November 3, 1988. From the Departments of Surgery, Clinical Pharmacology, and Medical Physics, Hope Hospital, University of Manchester School of Medicine, Salford M6 8HD, United Kingdom. The authors thank Hoffmann La Roche for performing the ascorbic acid analyses. The study was supported by grants from the Sheila Dawber Fund, the North Western Regional Health Authority and Hoffmann La Roche. Address for reprint requests: Professor M.H. Irving, Department of Surgery, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford M6 8HD, United Kingdom.
led to recommendations that such patients should receive ascorbic acid supplements (3, 4). It has also been reported that patients with fistulizing Crohn's disease have lower plasma, leukocyte, and intestinal ascorbic acid levels than patients without fistulas (1, 2). This finding raises the suggestion that fistula formation in Crohn's disease may result from impaired collagen synthesis (2, 5). If this were the case, and if it could be shown that ascorbic acid absorption is impaired in Crohn's disease, then the possibility of preventing fistula formation by giving ascorbic acid supplements would be worthy of consideration. Because ascorbic acid absorption and total body pools have not previously been investigated in patients with Crohn's disease, we have studied them in patients with fistulizing and nonfistulizing disease and in
Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
0163-2116/89/0400-055956.00/09 1989PlenumPublishingCorporation
559
PETTIT ET AL controls, using the absorption of orally administered L-[carboxyl-~4C]ascorbic acid. M A T E R I A L S AND METHODS Patients and Study Design. Patients undergoing operation for Crohn's disease were chosen for study as we wished to investigate the distribution of absorbed L[carboxyl-14C]ascorbicacid and its metabolic products in resected specimens of intestine using autoradiography and scintillation counting. It was felt that this might also provide information on the role of asCorbate in the pathogenesis of fistula formation in Crohn's disease (6). This assumption proved to be incorrect, and this aspect of the !nvestigation will not be considered in detail. The study was undertaken on 18 patients undergoing operation, six w~th fistulizing Crohn's disease, six with Crohn's disease without fistulas, and six controls. Clinical details of these patients are given in Table 1. Each study comprised the oral administration of an aliquot of L-[carboxyl-14C]ascorbic acid, followed by a period of equilibration. The patient then underwent operation, followed by a washout phase when the 14C was flushed from the body using large doses of unlabeled ascorbic acid. Each patient's ascorbic acid absorption was assessed using their total Cumulative urinary 14C excretion. In order to correct for any labeled ascorbate excreted in the breath, exhaled 14CO2 was measured. Total body pools of ascorbic acid were derived using the plasma 14C decay curve. The total dose of radioactivity received by each patient was less than 0.06 millisieverts. The study was approved by the Salford Health Authority Ethical Committee. Ascorbic acid intake was assessed using dietary history and food composition tables (7). Patients who were cigarette smokers were asked to abstain for the duration of the study as this habit has been associated with low ascorbate levels (8). Patients with Crohn's disease had the severity of their condition assessed using a disease activity scoring system (9). Each study extended from the day of admission to hospital (day 1) to the day of discharge. From day 1 until operation dietary intake was restricted to fluids that had a negligible ascorbic acid content. These comprised water, tea, coffee, and clear soup. On day 1 baseline samples of plasma, urine, and exhaled CO2 were collected for scintillation counting of background radioactivity. Administration of L-[carboxyl-14C]Ascorbic Acid and Subsequent Washout. An aliquot of L-[carboxyl14C]ascorbic acid containing 0.19-0.40 MBq of 14C was dissolved in 100 ml of water containing 50 mg of unlabeled ascorbic acid to act as a carrier; 1 ml was removed for scintillation counting, and the remainder was drunk by the patient (time 0 min). Twenty-four-hour urine collections were then commenced and continued until the end of each study. Each patient underwent operation two days after ingesting the labeled ascorbic acid. After operation, subjects received an intravenous injection of 500 mg ascorbic acid every 6 hr until unlimited oral fluids were allowed, at which time 1 g of ascorbic acid was given orally every 6 hr until the end of the study.
560
Sample Collection for Scintillation Counting of 14C Content. Plasma samples for scintillation counting were collected every 15 min for 3 hr after ingestion of the labeled ascorbic acid. Thereafter, they were collected every 30 min for 2 hr, hourly for 5 hr, twice daily for three days, and then once per day until the end of the study. An aliquot of each patient's daily 24-hr urine collection was removed for scintillation counting. Samples of exhaled CO2 were collected for scintillation counting 30, 60, 90, and 120 min after ingestion of the labeled ascorbic acid. Each sample was collected by bubbling air through trapping solution, comprising 1 ml of 1.0 M hyamine hydroxide in methanol, 1 ml methanol, and 10 p~l of 1% phenolphthalein, until the color of the indicator had just changed from pink to colorless. Scintillation counting was performed on a Packard Tri-Carb 4530 liquid scintillation counter using Insta-Gel for plasma samples, Pico-Fluor 15 for urine samples, and toluene scintillator for CO 2 samples. Sample Collection for Ascorbic Acid Analysis. Plasma and leukocyte samples were collected from each patient for ascorbic acid analysis every morning of the study. An aliquot of each patient's daily 24-hr urine collection was removed for ascorbic acid analysis. All samples were stabilized by mixing with metaphosphoric acid. The samples were stored at - 7 0 ~ C until analyzed. Leukocyte samples for ascorbic acid analysis were prepared using a modification of the method of Denson and Bowers (10): 4 ml of venous blood was added to 15 ml of leukocyte diluent and allowed to stand for 30 min while the erythrocytes sedimented. Then, 10 ml of supernatant was removed and the leukocyte content of a 20-~zl aliquot counted. The sample was centrifuged at 3000 rpm for 15 rain and the supernatant discarded by inverting the tube. The compacted button of leukocytes was then dissolved in 5% metaphosphoric acid. Ascorbic acid analysis was performed by a microfluorometric technique (11). Statistical Methods. Statistical comparisons were made using Wilcoxon's rank-sum test.
RESULTS Scintillation C o u n t i n g . P l o t s s h o w i n g e a c h group's mean cumulative urinary output of 14C are shown in Figure 1. The three plots are similar and differences between each g r o u p ' s daily urinary 14C output do not reach statistical significance on any day of the study. In each group a low urinary output of 14C was found during the equilibration period before operation. An increase in the urinary ~4C output occurred after operation as it was flushed from the patient by unlabeled a s c o r b i c acid. Towards the end of the studies, the urinary ~4C output diminished to negligible levels with the urinary output curves approaching horizontal, showing that virtually all the 14C had been flushed out of the patients. The total cumulative urinary outputs Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
A S C O R B A T E A B S O R P T I O N IN C R O H N ' S D I S E A S E TABLE 1. DETAILS OF PATIENTS UNDERGOINGL-[CARBOXYL-I4C]AsCORBIC ACID STUDIES
Initials
Age (years)
Sex
Weight/ Height 2 (kg/m 2)
Disease activity score
Ascorbic acid intake (mg/24 hr)
Drug treatment up to the time o f operation
Smoking habits
Patients with Crohn's disease and fistulae (group 1)
Type of fistula
N.C.
25
M
18.42
130
20
B.D.
59
M
23.78
187
30
M.Mc.N. D.P.
33 18
M M
22.41 17.71
137 129
93 26
D.R.
31
M
20.22
183
46
M.S.
57
F
19.10
162
363
Mean 37.2 20.27 155 SEM 6.93 0.97 10.8 Group 1 P > 0.9 P > 0.9 P = 0.02 vs 2 Patients with Crohn's disease without fistulae (group 2) F
18.0
94
100
B.F.
35
F
18.65
91
69
C.H.
24
F
18.55
144
46
L.!.
33
F
23.83
77
496
B.T.
43
F
19.65
99
C.T.
29
F
22.23
69
96 10.7
9.5 56
Ileocolocutaneous Colovesical
20-40 cigarettes/ day 20 cigarettes/ day
Ileocutaneous
Ileovaginal
Prednisolone 17.5 rag/day, azathio- prine 100 mg/day Prednisolone 7.5 mg/day, sulphasalazine 3 g/day Prednisolone 20 rag/day, sulphasalazine 3 g/day Prednisolone 30 rag/day Prednisolone 10 rag/day Prednisolone 15 rag/day, azaothioprine 100 mg/day
15 cigarettes/ day
Ileal
10 cigarettes/ day
Ileocecal
10 cigarettes/ day
lleocecal
15-20 cigarettes/ day 15 cigarettes/ day 15 cigarettes/ day
Jejunal Ileal Ileal
Operation performed
49
F
20.67
20
H.C.
59
F
23.12
30
R.D.
30
M
28.26
26
G.L.
54
M
26.47
40
F F
25.59 26.58 25.12 1.12 P = 0.02
155 50 54 20.8 P > 0.8
52 34 46.3 4.76 P > 0.4
Nonsmoker Occasional cigarette
Coloileocutaneous
129 74.3 P > 0.2
R.B.
P.L. D.S. Mean SEM Group 3 vs 1
Ileoileal
Site of disease
31
20.17 0.95 P = 0.02
15-20 cigarettes/ day Nonsmoker
96 54.4 P > 0.5
T.A.
Mean 32.5 SEM 2.60 Group 2 P > 0.05 vs 3 Control patients (group 3)
Prednisolone 15 rag/day Sulphasalazine 2 g/day, metronidazole 1.2 g/day None Prednisolone 20 mg/day, sulphasalazine 3 g/day Prednisolone 10 rag/day, sulphasalazine 2 g/day Prednisolone 15 rag/day
Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
Ketoprofen 200 rag/day None Cimetidine 800 rag/day Cimetidine 1600 rag/day None None
20 cigarettes/ day 10 cigarettes/ day 20 cigarettes/ day Nonsmoker Nonsmoker Nonsmoker
Abdominal Nissen fundoplication Cholecystectomy Highly selective vagotomy Transthoracic Nissen fundoplication Cholecystectomy Cholecystectomy
561
PETTIT
ET AL
100-% of 90 -dose given 80 ~
~
% of dose 1.4
Contro~
/
o f t 4C/litr e
of plasma
1.2
/
x~\
~x\
/
0.8
I
o.6
i
~,,
~'~-
.//
40_
ao --
,~/./
~ - ~ _ ? - ~ - _ C r o h n ' s without fistulae ~ "~Crohn's with fistulae
J,
20--
~ Days
0
Fig 1. Mean cumulative urinary ]4C output of the three groups of patients.
of ]4C, expressed as a percentage of the dose of ~4C ingested, are shown in Table 2. Graphs showing the mean plasma ~4C levels of each group plotted against time also have similar characteristics (Figure 2). Differences between each group's plasma 14C levels do not reach statistical significance at any time during the study. In every instance the plasma ~4C concentration rose to reach a peak 1-3 Y2 hr after ingestion of the labeled ascorbic acid subsequently falling to become almost stable in the period before operation. This indicated that the labeled ascorbic acid had become distributed throughout the patients' body tissues. After operation, unlabeled ascorbic acid was given to flush the 14C out of the patient. In every case, the plasma ~4C fell to approach background levels by the end of the study, indicating that the 14C had been completely flushed from the patients' body tissues. The peak plasma 14C concentration varied from patient to patient. The five smallest peaks, to 1% or less of the dose given per liter of plasma, all occurred in patients (B.D., D.R., C.H., B.T. and
1
2
3
4
5
d
7
8
9
10
Hours after giving 14C
Fig 2. Mean plasma 14C levels plotted against time for the three groups of patients.
H.C.) who had very low levels of plasma ascorbic acid (0.03-0.08 mg/100 ml) at the beginning of the study (Table 2). It seems likely that in these patients the labeled ascorbic acid was rapidly transferred from the plasma into depleted tissues, thereby preventing high plasma levels from occurring. In 11 of the 15 patients, a transient rise in plasma ]4C occurred at the beginning of the washout phase. This indicates that a redistribution of labeled ascorbic acid had occurred, it being transferred from a storage compartment into the plasma, before being excreted in the urine. Scintillation counting of the ]4C content of exhaled CO2 showed that less than 1% of the dose of ingested L-[carboxyl-14C]ascorbic acid was decarboxylated and excreted as expired 14CO2 in each patient. The results of scintillation counting of specimens from two patients (P.L. and L.I.) could not be analyzed, as problems were encountered when measuring their total ingested doses of 14C. The
TABLE 2, CUMULATIVE URINARY OUTPUTS OF 14C IN PATIENTS WITH CROHN'S DISEASE AND CONTROLS
Patients with Crohn's disease and fistulae (group 1) Urinary
outpMt of 14C Initials N.Co
B.D. M.Mc.N. D.P.
D.R. M.S.
Mean SEM
(% of dose given) 46.5 53.6 101.7 87.5 77.7 76.4 73.9 8.45
Plasma ascorbic acid beforeoperation (mg/lO0 ml) 0.38 0.08 0.85 0.45 0.07 1.27
Group 1 vs Group 2 P > 0.9
562
Patients with Crohn's disease without fistulae (group 2) Urinary
output of 14C lnitials T.A. B.F. C.H. L.I. B.T. C.T.
(% of dose given)
Plasma ascorbic acid beforeoperation (mg/lO0 ml)
55.4 90.8 46.6 -63.2 108.2 72.8 11.53 Group 2 vs Group 3 P >
0.91 0.28 0.03 0.56 0.07 0.48
0.8
Control patients (group 3)
Initials R.B.
H.C.
R.D. G.L. P.L. D.S.
Urinary output of 14C (% of dose given)
Plasma ascorbic acid before operation (mg/lO0 ml)
68.3 90.2 97.8 90.6 -54.6 80.3 8.11 Group 3 vs Group 1 P >
0.23 0.07 0.71 0.72 0.87 0.42
0.5
Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
A S C O R B A T E A B S O R P T I O N IN C R O H N ' S D I S E A S E ~ 30--
-- 3.0~ ./O
Operation
2.5-~ 2.0--
/
"\,
4/
\
Controls
i
! ,y
s
with fistulae
I
I 1
~.
f
./
_" /
/\.
\ /' "1( ,)<.
/
/l~
--.
0/.
~
Controls
....jo
/ \
~,~
"Ne Crohn's "with fistulae
s ~<~.
without fistulae
~ 10-
0.5-- X--- -..X. . . . . 0
/
I
20-
~ 158
~ 1.5~ 1.0--
^ uperation
~ 25-
5I 2
I 3
I 4
I 5
I 6
I 7
I 8
I 9
O--
I
I 1
Days
Fig 3. Mean daily plasma ascorbic acid levels of patients undergoing L-[carboxyIJ4C]ascorbic acid studies.
results of scintillation counting of all plasma samples from one other patient (N.C.) were disregarded because a white precipitate formed in the plasma that prevented the accurate measurement of emitted light. Ascorbic Acid Levels. In order to assess the reproducibility of the ascorbic acid assay, 17 plasma samples were divided into two aliquots before stabilization. The results of the standards (0.71 --- 0.12 mg/100 ml) and duplicates (0.69 --- 0.11 mg/100 ml) were very similar (P > 0.9) and differed by less than 3%. The mean daily plasma ascorbic acid levels of the three groups of patients are shown in Figure 3. The mean levels are similar in the three groups before operation and show little day-to-day variation. When the cold ascorbic acid was given to flush the 14C into the urine, plasma ascorbic acid rose in all three groups of patients. The differences between the plasma ascorbic acid levels of the three groups do not reach statistical significance on any day of the study. The mean daily leukocyte ascorbic acid levels of the three groups of patients are shown in Figure 4. Their levels rose after operation. Differences between the leukocyte ascorbic acid levels of the three groups do not reach statistical significance on any day of the study. The mean daily 24-hr urine ascorbic acid outputs of the three groups of patients are shown in Figure 5. The mean urinary ascorbic acid output was very small before operation in all three groups. After operation, they all exhibited a similar pattern of changes. Initially there was a rise in mean urinary ascorbic acid output. After reaching a peak, the mean levels fell before rising again at the end of the study. The patients' body pools of ascorbic acid, derived using scintillation counting and ascorbic acid results, are shown in Table 3. Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
i 2
I 3
I 4
I 5
I 6
I 7
I 8
I 9
Days
Fig 4. Mean daily leukocyte ascorbic acid levels of patients undergoing L-[carboxyIJ4C]ascorbic acid studies.
DISCUSSION The three groups of patients were reasonably well matched for age and dietary ascorbic acid intake (Table 1). Weight/height2, which is given as an index of body fatness, was significantly greater in the controls than in either group of patients with Crohn's disease (Table 1). This suggests either that the controls were overweight or that the patients with Crohn's disease had some nutritional impairment. The former seems more likely as gallbladder disease and gastroesophageal reflux are related to overweight status. None of the patients with Crohn's disease were clinically malnourished. The total cumulative urinary output of 14C represents the proportion of the oral dose of L[carboxylJ4C]ascorbic acid that was absorbed, assuming all of it has been flushed from the patients' body tissues during the washout phase of the studies and that the kidneys are the only route of elimination. Analysis of the exhaled air samples of CO: showed that less than 1% of the labeled ascor-
i
1400 ]
Operation ~ I t
1
/,
<,,:.,
i,oooI
/, ,".d.;.,;,,
9
,
,oo-
6oo-
I ~/o /
cr "\
/ , /I/ " '~r I ' / /
400-
/
,, ,,. ,, \ . / o \, ~-.
YV/
X
:,o
with fistulae "\. ~ .,.-.,0 ,, //Crohn's
"")<~without fistulae
/ /t
200 -
o- - - - ~ - - ~ 0
1 1
I 2
I 3
I 4
I 5
I 6
I 7
I 8
I 9
Days
Fig 5. Mean daily urine ascorbic acid outputs of patients undergoing L-[carboxyIJ4C]ascorbic acid studies.
563
PETTIT ET AL TABLE 3. TOTAL BODY POOLS OF ASCORBICACID WITH DATA USED FOR THEIR CALCULATION
Leukocyte
Total
F
ascorbic
Initials
Weight (kg)
Ascorbic acid intake (mg/24 hr)
acid
Plasma
Steady
body pools of ascorbic acid (mg/kg)
before
ascorbic
cumulative
state
operation (txg/lO8 WBC)
acid before operation (mg/lO0ml)
Ingested dose of 14C (dpm >( 10 7)
urinary recovery ofl4C)
plasma HC (dpm • 103/100 ml)
0.08 0.85 0.45 0.07 1.27
2.2861 1.1747 1.6576 2.0416 1.7486
0.5360 1.0170 0.8751 0.7773 0.7639
2.4593 9.2770 4.5567 2.4879 8.6901
4.88 14.38 23.42 6.29 36.72 17.1 5.91 P>0.4
0.91 0.28 0.03 0.07 0.48
1.9432 1.1658 2.4145 2.1183 1.4626
0.5542 0.9077 0.4655 0.6323 1.0822
10.7583 7.1944 4.7483 4.3906 7.8583
19.03 6.93 0.96 4.15 17.09 9.6 3.58 P>0.4
0.23 0.07 0.71 0.72 0.42
1.9387 1.7993 2.0840 2.0099 2.0904
0.6833 0.9020 0.9780 0.9060 0.5460
7.9053 3.9099 6.7008 9.0143 8.5017
Patients with Crohn's disease and fistulae (group 1) B.D. 76.2 30 7.7 McMc.N. 72.6 93 38.9 D.P. 59.3 26 11.7 D.R. 64.8 46 10.1 M.S. 52.0 363 15.4 Mean SEM Group 1 vs 2 Patients with Crohn's disease without fistulae (group 2) T.A. 46.3 100 12.1 B.F. 52.0 69 10.6 C,H. 49.9 46 10.4 B,T. 46.0 9.5 10.4 C,T. 53.4 40 14.7 Mean SEM Group 2 vs 3 Control patients (group 3) R.B. 46.5 20 4.0 H.C. 57.0 30 5.6 R.D. 76.0 26 9.0 G, L. 78.3 40 12.4 D.S. 60.6 50 17.7 Mean SEM Group 3 vs 1
bic acid had been decarboxylated and excreted as 14CO2. Additionally, it has been shown that less than 1% of an intravenous dose of L-[carboxyl~4C]ascorbic acid is lost in the feces over 10 days (12). Absorption of L-[carboxyl-14C]ascorbic acid based on the total cumulative urinary 14C output is similar for patients with fistulizing Crohn's disease, those without fistulas, and controls (Table 2). These results are similar to those previously found in healthy volunteers (13, 14), suggesting that ascorbic acid absorption is not impaired in Crohn's disease. In order to estimate the patients' total body pool of ascorbic acid from their plasma 14C decay curves, several assumptions have to be made. First, none of the labeled ascorbic acid is excreted by the liver before it reaches the systemic circulation. The failure of patients to excrete significant 14C in their feces after an intravenous injection of L-[carboxyl14C]ascorbic acid (12) would support this assumption. Second, complete equilibration of the L[carboxyl-lnC]ascorbic acid occurred before the patients underwent operation. The near horizontal
564
(= the total
7.45 4.31 27.77 18.00 8.85 13.3 4.28 P>0.8
plasma 14C decay curves at this time indicate that this assumption is valid. Third, plasma ascorbic acid and total body pool of ascorbic acid were in equilibrium before operation, an assumption supported by the fact that the daily plasma ascorbic acid levels at this time were stable in every patient. Given these assumptions, then the ratio of the steady-state plasma 14C concentration to the steady-state plasma ascorbic acid concentration before operation, will be the same as the ratio of total body 14C to total body ascorbic acid. All these variables are known except for total body pool of ascorbic acid, and this can be calculated (see Appendix). The calculated total body pools of ascorbic acid of the three groups of patients (Table 3) are not significantly different. Ascorbic acid total body pools have previously been studied in healthy volunteers (12-17). The values found are similar to those in both the controls and patients with Crohn's disease in the present study. The patients' daily plasma and leukocyte ascorbic acid levels and their 24-hr urine ascorbic acid Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
ASCORBATE ABSORPTION IN CROHN'S DISEASE outputs also provide information about ascorbic acid status. Patients depleted of ascorbic acid would be expected to have low plasma and leukocyte ascorbic acid levels and low urinary ascorbic acid outputs at the beginning of the studies. They would also be expected to retain a proportion of the ascorbic acid used in the washout phase of the studies until their body pools were replete. There were no statistically significant differences between the mean daily plasma ascorbic acid levels (Figure 3), leukocyte ascorbic acid levels (Figure 4), or urine ascorbic acid outputs (Figure 5) of the three groups of patients before operation, indicating that their total body pools of ascorbic acid were similar. The results of scintillation counting and autoradiography on resected specimens of intestine failed to show differences between patients with and without fistulas. The majority of the 14C was found within the intestinal blood vessels (two days after L-[carboxyl-14C]ascorbic acid ingestion), although some ~4C had migrated into the submucosa. More ~4C was present within the submucosa than in the underlying muscle, reflecting either increased mucosal vascularity or the higher submucosal collagen content. As a result of previous studies (19), we had expected to find differences in the amount of ~4C around fissure ulcers, in patients with and without fistulas, which might have provided insight into the pathogenesis of fistula formation. However little ~4C was found around fissure ulcers in autoradiographs from either group. This may have been because the time chosen for operation (two days after ~4C ingestion) was not optimal to demonstrate such differences. The present study has shown that ascorbic acid absorption and total body pools of ascorbic acid are similar in patients with Crohn's disease with and without fistulas and in the controls, the latter being symptomatic patients undergoing operation for gallbladder disease, gastroesophageal reflux, and peptic ulceration. It has not confirmed previous reports of patients with Crohn's disease having lower levels of plasma and leukocyte ascorbic acid than controls (1-4). This is probably because different (healthy) controls were used in the previous studies. The controls used in previous studies were healthy hospital workers and volunteers who would be expected to have normal levels of ascorbic acid. Many illnesses are associated with low levels of plasma ascorbic acid including myocardial infarction and acute infection (18). The results of the previous studies (1-4) may have therefore arisen Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
because the patients studied with Crohn's disease were ill and had low ascorbate levels because they were sick, rather than because Crohn's disease itself is associated with particularly low levels of ascorbate. This is supported by the results of the present study where more appropriate (nonhealthy) controls were chosen who proved to have similar levels of ascorbate as the patients with Crohn's disease. Our controls were hospitalized patients with symptomatic gastrointestinal disorders undergoing operation who were subject to the same stresses as the patients with Crohn's disease. Another factor explaining the discrepancy between the results of our study and the previous studies (1-4) also relates to the different types of controls chosen. In the previous studies the majority of healthy controls had high dietary ascorbate intakes, well above the minimum daily requirement (MDR) of 60 mg ascorbic acid per day. In contrast, 80% of our nonhealthy controls had dietary intakes below the MDR, thereby contributing to their lower levels of ascorbate. Their ascorbate intakes were comparable with those of our Crohn's patients, 60% of whom had ascorbate intakes below the MDR. A similar mechanism may explain reports of patients with Crohn's fistulas having lower ascorbic acid levels than patients with Crohn's disease without fistulas (1, 2). In a separate study of 70 patients with Crohn's disease (6), a negative correlation was found between Crohn's disease activity scores and plasma ascorbic acid levels (R = -0.22), showing a tendency for sick patients to have low levels of ascorbate. In the present study the two sickest Crohn's patients in each group, as indicated by their Crohn's disease activity scores (Table 1) (B.D. and D.R. with fistulas, C.H. and B.T. without fistulas) had the lowest plasma ascorbate levels and ascorbate body pools in their groups (Table 3), probably reflecting the severity of their illnesses. The patients with fistulas in previous reports (1, 2) may have had lower levels of ascorbate than the patients without fistulas because they were sicker. This seems likely as sepsis is often associated with fistula formation. Unfortunately their Crohn's disease activities were not scored, and so an informed conclusion cannot be made. Although our results have shown that ascorbic acid absorption is normal in patients with Crohn's disease, 60% of the patients studied received less than the MDR for ascorbic acid (60 mg/day). Therefore, an assessment of ascorbate intake should be made in all patients with Crohn's disease and ascor-
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PETTIT ET AL
bate supplements given where necessary. As ascorbic acid absorption and body pools are similar in patients with Crohn's disease with and without fistulas, there is little justification for a trial of ascorbic acid supplements to prevent fistula formation in Crohn's disease.
APPENDIX Method Used for Calculation of Total Body Pool of Ascorbic Acid
plasma [14C] plasma [ascorbic acid]
dose 14C x F body pool of ascorbic acid + (carrier dose • F)
where F is (total cumulative urinary output of 14C in dpm/ingested dose of 14C in dpm); plasma [14C] is steady-state plasma 14C concentration before operation in dpm/100 ml; plasma [ascorbic acid] is steady-state plasma ascorbic acid concentration before operation in mg/100 ml; dose 14C is ingested oral dose of a4C in dpm; body pool of ascorbic is total body pool of ascorbic acid in mg; carrier dose = 50 mg (the carrier dose of cold ascorbic acid given with labeled ascorbic acid); and body pool of ascorbic acid is (dose 14C x F x plasma [ascorbic acid]/plasma [14C]) - (carrier dose x F).
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Digestive Diseases and Sciences, Vol. 34, No. 4 (April 1989)
