Effect of Pancreatic Enzymes on Zinc Absorption in Cystic Fibrosis

Background: The trace metal zinc has a wide range of important physiologic roles. Indirect evidence suggests that fat malabsorption is associated with malabsorption of zinc. The objective of this study was to evaluate the effect of pancreatic enzyme replacement on zinc absorption in children and adolescents with cystic fibrosis.

Cited Here... Subjects were four boys and four girls ranging in age from 7 to 17 years of age. All were pancreatic insufficient. Stable isotope labels, ⁷⁰Zn and⁶⁷ Zn, were administered orally in divided doses on consecutive days with meals. Meals were identical on the first 2 study days. Subjects were randomized to have pancreatic enzyme replacement withheld on the first or second day. All fecal samples were collected quantitatively for 10 days after label administration and were analyzed individually for total zinc and isotopic enrichment using atomic absorption spectrophotometry and fast atom bombardment mass spectrometry, respectively. Fractional absorption of zinc was calculated from cumulative fecal excretion of unabsorbed label.

Cited Here... Fractional absorption while receiving enzymes was 0.50 ± 0.29 versus 0.38 ± 0.24 while not taking enzymes (p = 0.05).

Conclusions: These results indicate that fractional absorption of zinc is impaired by pancreatic insufficiency in patients with cystic fibrosis, and is improved by exocrine pancreatic enzyme replacement.

The trace metal zinc has a wide range of physiologic roles, and an adequate supply of this micronutrient is important during growth and development. Cases of severe zinc deficiency have been reported occasionally in association with cystic fibrosis, and there is some evidence to suggest that milder zinc deficiency also occurs ^(1,2). Abnormal zinc absorption has been suggested in other disease states associated with steatorrhea ^(3,4). It has been postulated that the zinc present in the lumen of the gut forms insoluble complexes with fats and phosphorus, as has been demonstrated in vitro with calcium and magnesium^(5,6).

If fat malabsorption does, in turn, impair the absorption of dietary zinc, withholding of pancreatic enzyme replacement therapy in cystic fibrosis patients with exocrine pancreatic insufficiency would have a negative effect on zinc absorption. The purpose of this study was to evaluate this hypothesis in children and adolescents with cystic fibrosis and exocrine pancreatic insufficiency.

MATERIALS AND METHODS

Fractional absorption of exogenous dietary zinc was determined by administering two different extrinsic zinc stable isotope labels on 2 consecutive days and determining cumulative fecal excretion of each label⁽⁷⁾. On 1 of the 2 days, normal pancreatic enzyme replacement therapy was withheld.

Subjects

Eight subjects (four boys and four girls) were recruited through the cystic fibrosis clinic at the Children's Hospital, Denver, Colorado. All subjects were studied at the time of admission for exacerbation of pulmonary disease. Subjects had clinically evident pancreatic insufficiency previously confirmed by fecal fat collections and were receiving exocrine pancreatic enzyme replacement therapy. Exclusion criteria included patients with previous history of significant abdominal surgery or resection, clinical or biochemical evidence of chronic liver disease or cholestasis, more than 15% of caloric intake via tube feedings, or treatment with corticosteroids at the time of admission. One subject taking corticosteroids chronically was maintained at a constant dose for the duration of the study. Subjects' age, sex, height and weight percentiles, calculated dietary zinc, plasma zinc, and study day they were not taking enzymes are given in Table 1. Informed consent was obtained at the time of admission for exacerbation of pulmonary disease and before initiation of the study in all subjects. The study was reviewed and approved by the Colorado Multiple Institutional Review Board.

Table 1

Diet

Information on recent dietary intake was obtained from the medical records, a 3-day diet record, food frequency questionnaire, and interview with the Pediatric General Clinical Research Center research nutritionist. Based on this history and in consultation with the individual subjects, an identical weighed diet was selected and prepared for the first 2 study days when the extrinsic zinc isotope labels were administered. Composition and weights of the meals for the remainder of the study period were also obtained and recorded. Any plate waste, which was minimal, was also weighed and recorded. Any zinc supplements were discontinued during the course of the study.

Pancreatic Enzyme Replacement Therapy

Each subject's routine pancreatic enzyme replacement was omitted on either day 1 or day 2 according to a table of random numbers. Exocrine pancreatic enzyme replacement remained constant for the remainder of the study.

Isotope Preparation and Administration

Enriched preparations of Zn stable isotopes (⁶⁷Zn and ⁷⁰Zn) were obtained from Oak Ridge National Laboratories (Oak Ridge, TN, U.S.A.). Accurately weighed quantities of the isotope were dissolved in 1 N H₂SO₄, diluted in triply deionized water, and titrated to pH 3. Each study subject received an accurately weighed quantity (≈1 mg total) each of stable zinc isotope label on 2 consecutive days: ⁷⁰Zn was given on day 1 and ⁶⁷Zn was given on day 2, divided equally between the three main meals of the day. This allowed measurement of zinc absorption over the course of a complete day for each isotope. Extrinsic tracers were administered in water at the midpoint of the meal.

Metabolic Collections

The study subjects were admitted to the Pediatric General Clinical Research Center for the duration of the metabolic study with the exception of two subjects who completed the final 4 days of their fecal collections at home. The study period was not commenced until subjects were afebrile and their appetite was no longer impaired by the acute exacerbation of their pulmonary disease. Weights were obtained and recorded daily using the same scale in the Pediatric General Clinical Research Center. All feces were collected in individual zinc-free plastic bags for a minimum of 8 days after the second isotope of zinc was administered.

Laboratory Procedures

Fecal samples were processed individually. Samples were homogenized and dried using a lyophilizer or drying oven until an accurate, stable weight was obtained. An aliquot was ashed at 425 °C for 24 hr, acid digested using concentrated HNO₃, then re-ashed at 425 °C for an additional 24-hr period. The ash was dissolved quantitatively in 6N HCl. The zinc concentration was determined by flame atomic absorption spectrophotometry, and total zinc in each stool sample was determined. The zinc was then separated from other trace metals and recovered using ion exchange resin columns⁽⁸⁾.

Zinc stable isotope ratios were determined by fast atom bombardment-induced secondary ion mass spectrophotometry on a double-focusing mass spectrometer(model VG 7070E HF; Fisons-VG Analytical, Manchester, U.K.) equipped with an Ion Tech (London, U.K.) atom gun ⁽⁸⁾. The presence of each isotope was determined relative to the natural abundance of ⁶⁶Zn. These ratios were then used to calculate the enrichment from the administered isotopes. Enrichment (ENR) is defined as: Equation

Equation 1A

Data Processing

Cumulative fecal excretion of orally administered ⁶⁷Zn and⁷⁰ Zn tracer was adjusted for absorbed tracer that had been secreted back into the lumen of the intestine and excreted ⁽⁷⁾. The fractional absorption of zinc (FAZ) is defined as:Equation

Equation 1B

Data Analysis

The effect of omitting pancreatic enzyme replacement therapy on fractional absorption of zinc was examined by paired-comparison Student's t tests.

RESULTS

Ten subjects were recruited, and eight completed the study. Two were excluded due to noncompliance with fecal collection. Plasma zinc concentrations ranged between 56 and 111 μg/dl as delineated inTable 1. For each subject, fractional absorption was lower when enzymes were withheld. Mean fractional absorption ± SD on and off enzyme therapy was 0.50 ± 0.29 and 0.38 ± 0.24, respectively. The mean difference (on enzymes minus off enzymes) was +0.12 ± 0.14(p = 0.05).

There was no correlation between fractional absorption on enzymes and either calculated dietary zinc intake or weight change during the study period.

DISCUSSION

The results of this study indicate that exocrine pancreatic enzyme replacement therapy in children and adolescents with cystic fibrosis and pancreatic insufficiency improves absorption of exogenous dietary zinc. This observation suggests that undigested and/or unabsorbed fat and/or protein interferes with zinc absorption.

If unabsorbed fat impairs absorption of exogenous dietary zinc, it is likely that it also decreases reabsorption of endogenous zinc that is secreted into the lumen of the small intestine in substantial quantities postprandially. A study design was considered, therefore, that would have permitted measurement of endogenous zinc losses in the feces and also measurements of metabolic balance for zinc as well as measuring fractional absorption of zinc. Such a study design would also have permitted comparison of fat absorption with and without enzyme replacement. However, this would have required an extended study of several weeks. Recruitment of older children and adolescents to such a study would have presented a formidable if not completely insurmountable challenge, and other variables may have changed substantially in this patient population during the course of a more prolonged study. It is also questionable whether pancreaticinsufficient children could tolerate a period of 2 to 3 weeks without pancreatic exocrine enzyme replacement therapy. Two of the considerable attractions of the study design selected were its acceptability and the close proximity of measurements when patients were and were not given enzymes, which resulted in a minimum of time for variables such as disease state or appetite to change significantly.

The results were notable for the wide interindividual range of values for fractional absorption, especially when receiving enzymes. Reliable interpretation of this wide range is not feasible without additional information, including data on fecal fat, zinc balance, and endogenous losses of zinc. It is perhaps worth noting, however, that the one subject with extremely high fractional absorption (0.85) also had an abnormally low plasma zinc concentration, suggesting impairment of zinc nutritional status, which may have accounted for the high absorption.

A study of young infants with cystic fibrosis who were diagnosed by the Colorado Universal Neonatal Screening Program at ≈2 months of age and before starting on pancreatic enzyme replacement therapy demonstrated unusually low fractional absorption of zinc and inappropriately high losses of endogenous zinc in the feces ^(9,10). As a result of these inappropriate losses, zinc balance was typically negative. These data, taken together with the results of this study, suggest that reabsorption of endogenous zinc may also be compromised by steatorrhea. The combination of events may lead to negative zinc balance. If this is so, it is reasonable to question why evidence of zinc deficiency in association with cystic fibrosis is not more apparent. This may be the result of effective pancreatic enzyme replacement therapy or it may be that tools for evaluating zinc status are not sufficiently sensitive. Most studies of zinc status in cystic fibrosis have relied solely on plasma or serum assays, which do not provide a sensitive index of zinc status. Even so, low circulating zinc levels have been reported in some studies of cystic fibrosis and have been linked to abnormalities such as growth retardation, impaired taste acuity, and low levels of retinol-binding protein and vitamin A ^(1,2).

The application of stable isotope techniques should allow further progress in our understanding of abnormalities of zinc status and metabolism in cystic fibrosis. Meanwhile, these results suggest that zinc supplementation should be considered in those cases that continue to have excessive fat excretion in the feces despite exocrine pancreatic enzyme replacement and in those pancreatic-insufficient children who have clinical features, such as growth retardation, that may be attributable at least in part to zinc deficiency.

Acknowledgment: This study was supported by grants RR00069, DK48520, and DK12432 from the National Institutes of Health, and by The Children's Hospital, Denver, Colorado, U.S.A.