Ovid: Bonham: Br J Surg, Volume 86(10).October 1999.1296-1301

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Volume 86(10), October 1999, pp 1296-1301

Early ascorbic acid depletion is related to the severity of acute pancreatitis [Original Article] Bonham, M. J. D.; Abu-Zidan, F. M.; Simovic, M. O.; Sluis, K. B.*; Wilkinson, A.*; Winterbourn, C. C.*; Windsor, J. A. Pancreatitis Research Group, Department of Surgery, Faculty of Medicine and Health Science, University of Auckland, Auckland and *Department of Pathology, Christchurch School of Medicine, Christchurch, New Zealand Correspondence to: Mr J. A. Windsor, Department of Surgery, Auckland Hospital, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand Presented to a meeting of the Australasian Surgical Research Society, Auckland, New Zealand, August 1995 and published in abstract form as Aust N Z J Surg 1996; 66: 243 Paper accepted 31 March 1999

Abstract Background: Ascorbic acid (AA) is an important endogenous antioxidant in plasma and has been shown to be decreased at the time of hospital admission in patients with acute pancreatitis. The aim of this study was to determine whether plasma AA concentration continues to decrease after admission and whether the extent of decrease is related to the severity of pancreatitis. Methods: Consecutive patients with mild (n = 62) and severe (n = 23) acute pancreatitis had plasma AA concentration measured on the day of recruitment and on days 2 and 5 by high-performance liquid chromatography. Results: The plasma AA concentration in patients with acute pancreatitis was significantly less than that in normal volunteers on days 0, 2 and 5 (P < 0.0001) and this was more marked in those with severe disease. There was a decrease in plasma AA concentration from day 0 to day 2 in patients with mild (P < 0.0001) and severe (P = 0.0005) pancreatitis, and from day 2 to day 5 in patients with severe pancreatitis (P = 0.023).

Conclusion: Endogenous plasma AA continues to decrease over the first 5 days in hospital and the extent is related to the severity of acute pancreatitis.

Introduction The management of acute pancreatitis is difficult because the key steps in its pathogenesis are not well understood. Experimental 1-4 and clinical 5,6 studies have provided some support for the concept that oxidative stress is the common pathway for the initiation of acute pancreatitis. Oxidative stress occurs when there is an imbalance between prooxidant activity and endogenous antioxidant defences. The most abundant endogenous antioxidant in the aqueous phase is ascorbic acid (AA) which is the bioactive form of vitamin C. Two studies of acute pancreatitis demonstrated that plasma AA was significantly below normal by the time patients were admitted to hospital, and that this fall was greater in acute pancreatitis than in other causes of acute abdomen 5,7. This study was undertaken to determine whether plasma AA concentration continues to decrease after admission and whether the extent of decrease is related to the severity of pancreatitis.

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Patients and methods Consecutive patients admitted to Auckland Hospital with a diagnosis of acute pancreatitis were recruited over an 18-month period. The diagnosis of acute pancreatitis was based on a serum amylase level greater than 1000 units/l (Phadebas; Pharmacia Diagnostics, Uppsala, Sweden; upper limit of normal 300 units/l) in association with typical abdominal pain. Written informed consent was obtained and the study was approved by the Northern Regional Health Authority Ethics Committee. Patients were excluded from this analysis if the pancreatitis was associated with pancreatic cancer or other major intercurrent illness. The patients were divided into mild and severe pancreatitis groups, based on their actual clinical outcome, as defined by the Atlanta classification 8 . The predicted severity of disease on admission was determined by the modified Glasgow criteria 9 , and the severity was monitored on a daily basis using the Acute Physiology And Chronic Health Evaluation (APACHE) II score 10 and the Organ Failure Score 11. Computed tomography (CT) was performed as indicated clinically. All patients received intravenous fluids. One patient with mild pancreatitis and six with severe pancreatitis received intravenous nutrition, which contained 0.6 mmol (100 mg) parenteral vitamin C per day, during the 5-day study. No patient received enteral nutrition during the study. Peripheral blood samples were taken from the fasting patients on recruitment (day 0) and on the morning of days 2 and 5. The blood was transported in heparinized tubes for immediate centrifugation and the plasma was stored at -80°C for subsequent analysis. AA was measured by high-pressure liquid chromatography (Walters 600E; Millipore, Medford, Massachusetts, USA) using a C18 reverse-phase column (octadecyl silane 5 µm, 220 × 4.6 mm) with an electro-chemical detector (Phillips Pye Unicam PU4022; Unicam, Cambridge, UK)12. This assay measures only reduced ascorbate. The smallest peaks measured were at least twice the average background fluctuation, and varied between 0.3 and 1.0 µmol/l from day to day. Concentrations below 1 µmol/l were classified as not detectable. Duplicates measured in the same run varied by 5 ± 4 per cent (n = 14). The intra-assay coefficient of variation for a plasma sample containing 60 µmol ascorbate was 3.9 per cent. The normal values for plasma AA were determined from 25 healthy volunteers (16 men, nine women; median age 32 (range 21-52) years) following an overnight fast. These volunteers were younger than the patients, but there is no evidence that plasma AA concentration varies significantly with age. Random urine samples were collected at the same time as the blood samples from 15 of the healthy volunteers and 17 of the patients to determine urinary AA loss. Serum and urine creatinine were measured to correct for dilution. The ratio of serum to urinary concentration was used as the dilution fraction. C-reactive protein (CRP) was measured by the hospital laboratory using a latex agglutination nephelometry method (Behring Diagnostics, Frankfurt, Germany). Statistical analysis was performed using Fisher's exact test for categorical data and the Mann-Whitney U test for continuous and ordinal data. The Mann-Whitney U test was used to compare the same time points between the two groups, and the non-parametric Wilcoxon matched-pairs signed rank test was used to compare different time points within the same group. P < 0.05 was accepted as significant.

Results There were 85 patients (44 men, 41 women; median age 56 range (16-96) years) recruited for this study, of whom 62 had mild and 23 severe pancreatitis.

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Six additional patients were excluded from this analysis because of pancreatic cancer (n = 4), biliary peritonitis (n = 1) and renal transplant rejection (n = 1). Twelve patients with mild acute pancreatitis were discharged and three with severe acute pancreatitis died within the 5-day study period. These patients did not have measurements on day 5. Patients were recruited a median of 7 (range-4 to 30) h after formal admission to hospital, excluding the five who were already inpatients. There were no significant differences between these patient groups for age, sex distribution or aetiology of pancreatitis (Table 1). Nine of the 23 patients with severe pancreatitis and one with mild disease were referred from another hospital, which accounts for the significantly longer duration of pain before admission in the severe group. When these patients were excluded there was no longer a significant difference (P = 0.77) in the interval from the initial onset of symptoms to recruitment into the study between mild and severe groups. The two patient groups were significantly different in terms of the predicted severity (as defined by the number of positive criteria from the modified Glasgow score), the actual severity of pancreatitis (APACHE II and Organ Failure Score) and plasma CRP level (Table 1).

Table 1 Patient demographics, aetiology, duration of pain and severity at time of recruitment Nineteen patients with severe pancreatitis and 17 with mild pancreatitis had CT. Patients with mild pancreatitis received intravenous fluids for a median of 3 (range 1-13) days compared with 21 (range 2-106) days for patients with severe pancreatitis. Patients with mild pancreatitis resumed eating at a median of 4 (range 1-8) days compared with 37 (range 3-103) days for the 16 patients who survived the attack of severe pancreatitis. The clinical outcome of the patients is summarized in Table 2. Seven patients with severe pancreatitis died. Two of the ten patients admitted to the intensive care unit (ICU) died, one after 38 days in the unit (following prolonged sepsis of multiple organ failure) and one later on the ward from pneumonia after 85 days in hospital. Four additional patients were not expected to survive and declined admission to the ICU. Three of these patients died from respiratory failure (days 2, 3 and 8) and the fourth patient died from multiple organ failure on day 10. The remaining death occurred on the ward on day 3 from a myocardial infarction in a patient with pre-existing unstable angina. Patients with severe pancreatitis stayed in hospital significantly longer and developed significantly more organ dysfunction than those with mild disease (Table 2).

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Table 2 Clinical outcome of patients with acute pancreatitis The median plasma AA concentration of the 25 healthy volunteers was 59 (range 27-120) µmol/l (Fig. 1). By comparison, the patients had persistently and significantly lower plasma AA concentrations on days 0, 2 and 5 (P < 0.0001). On days 2 and 5 the patients with severe pancreatitis had significantly lower plasma AA concentrations than those with mild pancreatitis (P < 0.001). There was no significant overall difference between mild and severe pancreatitis on day 0.

Fig. 1 Plasma ascorbic acid concentration on days 0, 2 and 5 in patients with mild and severe acute pancreatitis, and in healthy volunteers. Results are expressed as a box plot showing median (horizontal line) and interquartile range (box). The whiskers extend to the highest and lowest values within one and a half times the interquartile range from the median. *P < 0.0001; P < 0.001 (Mann-Whitney U test) There was a significant decrease in plasma AA concentration from day 0 to day 2 in patients with mild (P < 0.0001) and severe (P = 0.0005) pancreatitis. A further decrease occurred from day 2 to day 5 in patients with severe pancreatitis (P = 0.023) but not for mild pancreatitis (P = 0.85). These findings were not altered when eight patients with pancreatitis who were admitted from other institutions were excluded from the analysis. The plasma AA concentration was negatively correlated with CRP on days 0, 2 and 5 (P = 0.02, P = 0.002 and P = 0.004 respectively, Spearman's [rho]). The above analyses were also performed after excluding the 14 patients (six mild and eight severe) who presented more than 48 h after the onset of abdominal pain. There was no longer a

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significant difference between patients with mild and severe pancreatitis on days 0 (P = 0.9, Mann-Whitney U test) and 2 (P = 0.07, Mann-Whitney U test), although it was significant on day 5 ( P = 0.008, Mann-Whitney U test). Analysis of the trends over the 5 days revealed a significant decrease in plasma AA concentration from day 0 to 2 in patients with mild (P < 0.0001, Wilcoxon test) and severe (P < 0.005, Wilcoxon test) pancreatitis. A further significant decrease in plasma AA concentration occurred from day 2 to 5 in patients with severe pancreatitis (P = 0.017, Wilcoxon test) but not in mild pancreatitis (P = 0.8, Wilcoxon test). Urinary AA concentration was measured on days 0 and 5 in 17 patients (12 mild, five severe) and 15 healthy volunteers, and corrected for dilution by measurement of serum and urine creatinine (Table 3). There was no significant difference between healthy volunteers and patients with mild or severe pancreatitis. The corrected urine AA concentration correlated with the plasma AA concentration (P < 0.001, Spearman's [rho]).

Table 3 Urinary ascorbic acid concentration corrected by the ratio of urine to serum creatinine in healthy volunteers and patients with mild and severe pancreatitis

Discussion This study demonstrates that plasma AA concentration was significantly below normal in patients with acute pancreatitis at the time of admission to hospital, that these low levels continued to fall after admission and that the levels were lowest in patients with severe disease. After excluding patients with prolonged symptoms, the initial values were similar in mild and severe disease, although the decrease was greater by day 5 in patients with severe pancreatitis. The early events in the pathogenesis of acute pancreatitis are not well understood, but the uniformity of the histopathological appearances in the face of different aetiologies suggests a common pathway. Oxidative stress has been proposed as this pathway 1-5,13 and occurs when there is an increase in pro-oxidant activity in relation to endogenous antioxidant defences. AA is the bioactive form of vitamin C and a potent endogenous aqueous antioxidant which scavenges oxygen-derived free radicals produced by activated neutrophils and the hypoxanthine-xanthine oxidase system 14. The findings of this study support the proposal that significant oxidative stress occurs early in acute pancreatitis. Other evidence for early oxidative stress comes from the finding that manganese superoxide dismutase, a mitochondrial antioxidant enzyme and a marker of myocardial ischaemia-reperfusion injury 15, was significantly raised in serum of patients with

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severe acute pancreatitis 16. The pancreas is particularly prone to ischaemia 17 and it has been shown that patients with severe pancreatitis who had significant gastric intramucosal ischaemia, as measured by tonometry, were more likely to die 18. Scott et al.7 have reported low levels of plasma AA in patients with acute pancreatitis at the time of admission to hospital. They measured both plasma AA and total vitamin C and included a group of patients who were admitted with other causes of acute abdominal pain. The ratio of AA to vitamin C was strikingly different between normal subjects (80 per cent, n = 30), patients with acute abdominal pain (62 per cent, n = 27) and those with acute pancreatitis (less than 17 per cent, n = 29). This suggests that patients with acute pancreatitis are subject to greater oxidative stress. There are two other possible explanations for the depletion of plasma AA. Patients might have developed acute pancreatitis because of a pre-existing decreased concentration of plasma AA. There are no reasons to suspect this to be the case and the population study required to investigate this would be prohibitively large. Alternatively patients with acute pancreatitis might have increased urinary AA losses. Urinary vitamin C levels have been measured after surgery, but not following acute pancreatitis. Crandon et al.19 measured increased urinary vitamin C concentrations in nine patients after operation, but these patients had received intraoperative intravenous vitamin C. In contrast, Mason et al.20 found that 24-h urinary excretion of vitamin C was lowest on the first day after operation and that it returned to normal over 8 days. Vallance 21 suggested that the mean 33 per cent fall in plasma total vitamin C levels measured on the first day after major surgery was due to increased urinary excretion. The data derived from the measurement of urinary AA concentration in patients with mild and severe pancreatitis in the present study (Table 3) suggest that the depletion of plasma AA cannot be explained by increased urinary losses. The significant correlation indicates that those with higher serum levels tend to have higher urine levels. There is a wide range of urinary AA levels in patients and volunteers, which means that a type II error is possible with the small number of subjects. It is not possible to determine, from this or other studies, whether oxidative stress causes acute pancreatitis or whether it is an effect of it, predisposes to it or is just an epiphenomenon. Regardless of this, the fact that the profound depletion of plasma AA persists for some days suggests that a clinical trial investigating the impact of treatment with AA on oxidative stress and clinical outcome is justified 5

Acknowledgements This work was supported by the Auckland Medical Research Foundation, the Maurice & Phyllis Paykel Trust, and the Health Research Council of New Zealand. M.J.D.B. was supported by a Ruth Spencer Medical Research Fellowship. The authors thank Karen Silvers for performing the measurements of urinary ascorbic acid.

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