Prevention of recurrent pancreatitis in familial lipoprotein lipase deficiency with high-dose antioxidant therapy.

We describe a dramatic response to antioxidant therapy in three patients with familial lipoprotein lipase deficiency complicated by frequent severe episodes of pancreatitis who had failed to respond to other dietary and pharmacological measures. Antioxidant therapy may be an important advance in the management of this type of patient.

Deficient nifedipine oxidation: a rare inherited trait associated with cystic fibrosis kindreds.

Previous studies have indicated that there is weak genetic linkage between the defective gene in cystic fibrosis (CFTR) and the gene encoding the nifedipine metabolizing enzyme P4503A4 which are both located on chromosome 7. To examine further this possible association, nifedipine metabolism was investigated in a group of 59 volunteers, and 17 adult cystic fibrosis patients and 37 of their relatives. In agreement with the majority of previous studies, the volunteer group showed a unimodal distribution of recoveries for the major metabolite M-II ranging from 33 to 78% excretion in 8 h. In the case of both the cystic fibrosis patients and their parents, the distribution of recoveries was shifted to the left with five out of 20 parents and three out of 11 unrelated cystic fibrosis patients showing recoveries below the range observed in the volunteer group. This poor metabolism appeared to be both reproducible and heritable and did not appear to be a consequence of mutations in the CFTR gene.

Heightened free radical activity in pancreatitis.

Three markers of free radical oxidation of lipids--9 cis, 11 trans isomer of linoleic acid, conjugated dienes and ultraviolet fluorescence products--were measured in the phospholipid fraction of duodenal juice collected in the first 10 min after an intravenous injection of secretin. The volume of aspirate was similar in 11 controls and in 25 patients who had sustained an attack of pancreatitis 6 weeks earlier--acute pancreatitis (AP) 10, chronic pancreatitis (CP) 15. The concentration of each marker was very significantly higher in the patients; the output of the isomer gave the best discrimination from controls; and ultraviolet fluorescence products were substantially higher in the subgroup with CP than with AP. The serum % molar ratio of the isomer to linoleic acid was measured in 25 controls, 14 AP and 17 CP patients: the highest levels were found in the CP group. Heightened hepatic free radical activity involving lipid isomerization as well as lipid peroxidation pathways is a feature of pancreatitis--probably antedating the attack and persisting well after clinical recovery--the difference between CP and AP being in the degree of abnormality. We argue that these hepatic changes mirror changes in pancreatic-acinar cells and that increased free radical activity in both organs is due to a shortfall of antioxidants in the face of cytochromes P450 induction by xenobiotics. Therefore, a combination of preventive and chain-breaking antioxidants may be useful in preventing further attacks of pancreatitis and controlling background pain in chronic disease.

On the identification of a conjugated diene component of duodenal bile as 9Z,11E-octadecadienoic acid.

The lipid free radical marker, termed diene conjugation, in secretin-stimulated human bile obtained from the duodenum, was shown by high performance liquid chromatography and capillary gas chromatography-mass spectrometry, to be due mainly to 9Z,11E-octadecadienoic acid. The lack of evidence for possible conjugated diene isomers argues for an enzymatic origin of this product rather than being due to a random free radical mechanism, as is usually assumed.

The nature of diene conjugation in human serum, bile and duodenal juice.

Diene-conjugated lipids have been located by HPLC in serum, bile and duodenal juice. Whether esterified or not the same predominant fatty acid is responsible for most of the diene conjugation in all of these biological fluids. Initial attempts to generate this fatty acid in pure lipid by classical lipid peroxidation in vitro were unsuccessful. Ultraviolet irradiation of free fatty acids in the presence of protein produced diene-conjugated lipids similar to those found in vivo. The predominant diene-conjugated fatty acid in vivo is an isomerised C18:2 compound.

Antioxidant therapy for recurrent pancreatitis: biochemical profiles in a placebo-controlled trial.

The usefulness of micronutrient antioxidant therapy for recurrent (non-gallstone) pancreatitis has recently been endorsed by a 20-week double-blind double-dummy cross-over trial in 20 patients. Treatment was delivered as two types of tablets, providing daily doses of 600 micrograms organic selenium, 9000 i.u. beta-carotene, 0.54 g vitamin C, 270 i.u. vitamin E and 2 g methionine. We report antioxidant profiles in blood samples collected before entry, at the cross-over stage and upon completion of trial. Baseline serum concentrations of selenium, beta-carotene and vitamin E in the patients were significantly lower than in healthy controls, were unaltered by placebo and normalized by active treatment, but reverted to basal values in the subgroup that received placebo subsequently. The baseline serum concentration of a free radical marker--the 9-cis, 11-trans isomer of linoleic acid--was significantly higher in the patients than in controls, fell inexplicably in the placebo phase and fell further upon active treatment. Discriminant analysis eliminated the overlap in free radical marker and selenium concentrations between control sera on the one hand and baseline or post-placebo samples from the patients on the other: antioxidant treatment normalized the relationship between these biochemical parameters. Subnormal baseline serum levels of S-adenosylmethionine drifted downwards upon active treatment whereas a sharp rise was noted when a relapse of pancreatitis occurred during the placebo phase. The results confirm that adequate exposure to antioxidants in the active treatment phase was associated with amelioration of oxidative stress, and that there was no residual effect 10 weeks after switching over to placebo treatment. Furthermore, the paradoxical behaviour of S-adenosylmethionine may imply that the beneficial effect of micronutrient antioxidants in recurrent pancreatitis is linked with preservation of the methionine trans-sulfuration pathway in pancreatic acinar cells.

Antioxidant therapy for recurrent pancreatitis: placebo-controlled trial.

Oxidant stress has been proposed as the initiating pathogenetic mechanism in pancreatitis, hence micronutrient antioxidant therapy has been assessed in patients with recurrent attacks and/or constant pancreatic pain. In a 20-week double-blind double-dummy crossover trial active treatment was given as two types of tablets providing daily doses of 600 micrograms organic selenium, 9000 IU beta carotene, 0.54 g vitamin C, 270 IU vitamin E and 2 g methionine. Of 28 patients enrolled, 20 adhered to the full protocol (idiopathic chronic 8, alcoholic chronic 7, idiopathic acute 5). Six patients had an attack whilst on placebo but none whilst on active treatment (P = 0.032). Analysis of visual analogue scoresheets to compare background pain in the 10-week period before entry and during each phase of the trial, using a 10-cm scale for each of 11 best descriptors, endorsed the beneficial effect of active treatment (placebo v baseline, P = 0.073; active v baseline, P less than 0.001; active v placebo, P = 0.049). The same trend emerged from analysis of pain-score diaries by conventional and time series methods. Micronutrient antioxidant therapy thus offers a new approach to the treatment of recurrent (non-gallstone) pancreatitis and/or pancreatic pain.

Sclerosing cholangitis with hepatic microvesicular steatosis in cystic fibrosis and chronic pancreatitis.

The association between asymptomatic primary sclerosing cholangitis and exocrine pancreatic disease was underlined by the findings in a patient with cystic fibrosis and in another with chronic pancreatitis. In each case hepatocytes showed extensive microvesicular steatosis and studies of drug metabolism suggested hepatic enzyme induction: biliary or serum analysis, or both, disclosed raised concentrations of a lipid-based marker of free radical oxidation. These findings suggest that toxic metabolites of oxygen or other chemicals may have a role in the pathogenesis of the bile duct lesion.

Iron, ascorbate and copper status of Sowetan Blacks with calcific chronic pancreatitis.

Vitamin C can be used to overcome oxidative stress and ease pain in chronic pancreatitis. But its use is deprecated in conditions of tissue iron overload, because its bioactive form, ascorbate, can accelerate free-radical reactions that are driven by transition metals. We measured iron, ascorbate and copper in Sowetan Blacks (RSA) with chronic pancreatitis, obtaining serum/plasma from 14 consecutive patients and 15 controls. Compared with data from corresponding groups in Manchester, African samples had less ascorbate (p < 0.0001), but more caeruloplasmin (p < 0.0001). African and British controls had comparable iron and iron-binding capacity. Plasma from African patients had less ascorbate than that from African controls (p < 0.005) and in six samples, ferritin exceeded 300 micrograms/l (677 pmol/l). Low-molecular-mass iron or copper, capable of participating in free radical reactions, was not detected. British patients, had similar caeruloplasmin levels to African patients but higher ascorbate levels. There is no evidence of iron overload in our African samples. Outwardly healthy controls from Soweto have elevated levels of caeruloplasmin, possibly to compensate for dietary deficiency of ascorbate. Persistent oxidative stress is a unifying feature of chronic pancreatitis, but its degree is higher in African than British patients. Supplements of vitamin C should be safe in Blacks of southern Africa.

Recalcitrant pancreatitis: eventual control by antioxidants.

A woman, 61 years of age at her first attack of pancreatitis, had further attacks every few months during the next two years despite cholecystectomy for gall stones, and pancreato-duodenectomy for pancreas divisum and duodenal diverticulum. Since starting treatment with antioxidants, she has been free of attacks for two years.

Routes of protein secretion in the isolated perfused cat pancreas.

The routes by which secretory proteins leave the pancreas have been studied during single-pass perfusion of the isolated cat pancreas with a physiological salt solution. The amounts of amylase and total protein appearing in pancreatic juice, venous effluent, and the exudate escaping from the surface of the gland (probably representing lymph) were measured during stimulation with secretin alone and together with bolus injections of acetylcholine. During stimulation with secretin alone, the amylase output in venous effluent was four times greater than in pancreatic juice or exudate. In contrast, most protein appeared in exudate, some in the venous effluent, and a very small amount in pancreatic juice. Acetylcholine transiently increased the output of both amylase and total protein in pancreatic juice to values that greatly exceeded those in venous effluent and exudate. After a delay of 10 min, it also increased the output of amylase and total protein in exudate, but had no effect on venous effluent. In conclusion, secretory proteins enter the venous circulation at a constant rate (i.e., not influenced by acetylcholine stimulation). Some also appear in exudate (lymph), the proportion of which is determined by stimulation. Whether the proteins in these two fluids and in pancreatic juice are derived from the same intracellular pool remains to be determined.

Selenium and diabetes in the tropics.

We have examined the possibility that selenium deficiency may underlie one or more of the following peculiarities of chronic pancreatitis in tropical as compared to temperate zones: much higher prevalence, propensity for pancreatic calculi, and high frequency of diabetes. Selenium was measured by graphite furnace atomic absorption spectrometry in sera from 20 healthy volunteers, 36 patients with chronic pancreatitis (calcific 35, diabetic 32), and 23 patients with primary forms of diabetes, from Madras, South India; results were compared with data from 41 controls and 37 patients with chronic pancreatitis (calcific 13, diabetes 8) from Manchester, North West, England. We conclude that (a) bioavailability of selenium is equally high in each geographic area; (b) decrement in serum selenium (p less than 0.001) is of a similar order in Manchester and Madras patients, which denies a connection with calculi formation or pancreatic exocrine failure (since the incidence of these two problems was substantially higher in the Madras series); and (c) selenium levels do not account for accelerated course to diabetes in tropical chronic pancreatitis.

Altered clearance of sulphobromophthalein (BSP) in patients with pancreatic disease.

The components of the plasma disappearance curve of sulphobromophthalein--Ki, K2, K1--were analysed in 26 patients with pancreatic disease. The mean corrected initial disappearance rate constant, K1, in the patients significantly exceeded the published mean value in controls: all but four patients had a value equal to or higher than the upper limit of the reference range (mean +2 SD). The mean uncorrected initial disappearance rate constant, Ki, in the patients was not significantly different from the mean in controls but the mean of the second exponential, K2, was significantly reduced. At least one abnormality in the test (Ki, K2, K1) was present in 24 of the 26 patients studied (93%), although clinical evidence of hepatic dysfunction was generally unimpressive. The possible implications of the results are discussed with reference to previous studies.

An evaluation of the low-pH enzymatic assay of urinary D-glucaric acid, and its use as a marker of enzyme induction in exocrine pancreatic disease.

We have evaluated a low-pH enzymatic method for measuring urinary D-glucaric acid, and its usefulness as a marker of 'enzyme induction' in patients with exocrine pancreatic disease. The coefficient of variation lay between 7.5 and 10.9% for within-batch precision, and between 7.9 and 19.8% for between-batch precision. The useful range of the method was 20-200 mumol/l, with a lower detection limit of 11 mumol/l. The molar concentration ratio of D-glucaric acid to creatinine in urine correlated with the 8-h output of D-glucaric acid (p less than 0.005): both indices were significantly higher in a group of 29 patients with exocrine pancreatic disease than in controls (median ratios 4.6 and 2.9 X 10(-3), p less than 0.005; median outputs 14.0 and 8.8 mumol/8 h, respectively, p less than 0.005). Comparison with the results of theophylline tests in the same group of patients showed that whereas 72% of patients had theophylline clearances higher than the highest value in controls, 45% of the group had increased D-glucaric acid/creatinine ratios, whilst only 21% had increased outputs of D-glucaric acid. Paradoxically, in patients with established liver disease in whom drug metabolism was impaired urinary D-glucaric acid values were amongst the highest encountered in the study. Thus, the obvious advantages of the method--non-invasive, simple, reproducible, inexpensive, easily applied to out-patients--are offset by an unacceptably low predictive value as an indicator of microsomal 'enzyme induction'.

On optimising the diagnostic yield of secretin pancreozymin tests.

The results of 407 secretin-pancreozymin tests were analysed by a variety of statistical methods, in an attempt to optimize diagnostic yield. The best diagnostic yield accrued from selection of a point corresponding to 95% specificity and 60% sensitivity on each of two virtually superimposable receiver-operator curves--using either bicarbonate output or a discriminant function derived from multivariate analysis--and the anticipated yield approximated to that realised in a further prospective series of 150 cases. At a 25% hypothetical local prevalence of chronic pancreatic disease (including chronic pancreatitis and pancreatic cancer), the positive predictive value was 80%, the negative predictive value 88% and the efficiency was 86%--values that are not dissimilar to those reported in a study in which both hormones were given simultaneously by constant intravenous infusion for 105 min with multivariate analysis of the results. We conclude that (a) measurement of bicarbonate output in 30 min after an appropriate dose of secretin given as a bolus injection yields results that are comparable to those obtained when secretin and pancreozymin are given by constant intravenous infusion in doses to evoke maximal secretory responses; and (b) the yield of hormone tests using duodenal intubation is far from ideal.

Serum copper oxidase activity (coeruloplasmin) in chronic pancreatitis: inverse correlation with pancreatic exocrine function.

Serum copper oxidase activity (coeruloplasmin, ferroxidase, I, EC 1.16.3.1) and serum C-reactive protein (CRP) were estimated in 43 patients with chronic pancreatitis; total serum copper was also measured in 23 patients. Pancreatic exocrine function was assessed in all patients and compared with the respective copper oxidase activities. The following results emerged: (1) there was a strong positive linear relationship between total serum copper and serum copper oxidase activity; (2) in 34 patients CRP was undetectable Twenty-six patients were untreated and eight had received pancreatic extracts for variable periods of time. In the untreated patients there was an inverse correlation between serum copper oxidase activity and pancreatic exocrine function; (3) in the eight treated patients serum cooper oxidase activity was less than in untreated patients; (4) in three patients who were assessed before and six months after administration of pancreatic extracts serum copper oxidase activity showed a significant reduction with treatment; (5) nine patients with elevated CRP values also had raised serum copper oxidase activities but there was no correlation between these two serum constituents. The rise in serum copper which occurs in uncomplicated and untreated chronic pancreatitis does not represent an acute phase reaction. Our results provide further evidence that the pancreas assists in regulating copper metabolism in man.

Induction of cytochromes P-450 in pancreatic disease: consequence, coincidence or cause?

We have examined the pharmacokinetics of antipyrine and of theophylline--validated probes for cytochromes P-450 activities--in a series of patients with pancreatic disease. The half-life of each drug was significantly lower, and its clearance faster, in patients than in controls and this pattern was detected in the subgroups with acute pancreatitis (6), chronic pancreatitis (22), or pancreatic cancer (4). These data suggest induction of cytochromes P-450 in all forms of exocrine pancreatic disease. Enzyme induction is unlikely to be secondary to pancreatic malfunction since there was no correlation between prevailing exocrine status, as assessed by secretin-pancreozymin tests, and the half-life or clearance of either drug. The corollary is that induction of the mono-oxygenases by environmental agents, both recognised and unidentified, is a primary event in pancreatic disease. The possible relevance of this finding is discussed.

Micronutrient antioxidant status in black South Africans with chronic pancreatitis: opportunity for prophylaxis.

Biochemical assessments of micronutrient antioxidant status were done in 14 consecutive black patients with calcific chronic pancreatitis and 15 controls at Soweto, near Johannesburg in southern Africa. The patients showed subnormal levels of vitamin C in plasma; selenium, beta-carotene and alpha-tocopherol in serum; and inorganic sulphate (as an index of long-term sulphur amino acid intake) in urine (P < 0.001 for each): furthermore, among the patients ascorbate constituted a lower fraction of vitamin C (P < 0.002), indicating heightened oxidation of the bioactive form. By comparing the results in Sowetan controls with reference ranges from Manchester, UK, the markedly lower vitamin C and, hence, ascorbate levels in the Sowetans was underlined (P < 0.001) and their selenium levels were also lower (P < 0.001), but beta-carotene, alpha-tocopherol and inorganic sulphate levels were comparable. The very low bioavailability of ascorbate among Sowetan controls is reminiscent of our previous finding in outwardly healthy people at Madras in southern India: in both these areas chronic pancreatitis is currently endemic, has a propensity to pancreatic calculi and runs a virulent course towards premature death from diabetes, malnutrition or pancreatic cancer. Considering that low ascorbate levels are a feature in patients with chronic pancreatitis who develop pancreatic calculi at Manchester and that antioxidant supplements ameliorate painful symptoms, we suggest that poor antioxidant intake may predispose underprivileged tropical communities to the disease. If so, there could be an opportunity for prophylaxis through a daily tablet containing vitamin C, perhaps along with selenium at Soweto and beta-carotene at Madras.

Relationship between PABA and Lundh tests: lack of influence of duodenal pH in vivo.

Twenty-five patients with chronic pancreatitis and 25 patients with non-pancreatic abdominal disorders were investigated by Lundh and BT PABA/14C-PABA tests of pancreatic function. The following results emerged: (1) There was a strong positive linear relationship between mean trypsin activity (MTA) and mean chymotrypsin activity (MCA) in duodenal aspirates after a Lundh test meal. (2) There was a strong positive linear relationship between chymotrypsin activity measured with BTEE, or BT PABA as substrate. (3) There was a strong positive correlation between MTA, or MCA, in duodenal juice after a Lundh meal and urinary PABA recovery, or the PABA/14C excretion index (PEI) in patients with chronic pancreatitis, but not in controls. (4) There was no correlation between the pH of duodenal juice in Lundh tests and PABA recovery, or PEI, in patients with or without pancreatic disease. We conclude that the Lundh and BT PABA/14C-PABA tests are equally discriminatory methods of assessing pancreatic exocrine function. The rate limiting effect of pH on BT PABA hydrolysis reported in in-vitro studies does not affect the practical clinical value of the BT PABA/14C-PABA test.

Observations on the BT PABA/14C-PABA tubeless test of pancreatic function.

The diagnostic accuracy of the BT PABA/14C-PABA tubeless test of pancreatic function was assessed in a prospective study of 140 patients. Drug or isotopic interference invalidated 16 tests (11.4%). The sensitivity of the PABA/14C excretion index was 76.7% and the specificity 85.7% using the mean -2 SD value of 0.82 as the cut-off point; 74.4% sensitivity and 95.7% specificity using the mean -3 SD value of 0.76. The potential usefulness and limitations of this modern version of the PABA test are discussed.