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Gastrointestinal Outcomes and Confounders in Cystic Fibrosis

Borowitz, Drucy; Durie, Peter R; Clarke, Lane L; Werlin, Steven L; Taylor, Christopher J; Semler, John; De Lisle, Robert C; Lewindon, Peter; Lichtman, Steven M; Sinaasappel, Maarten; Baker, Robert D; Baker, Susan S; Verkade, Henkjan J; Lowe, Mark E; Stallings, Virginia A; Janghorbani, Morteza; Butler, Ross; Heubi, James

Journal of Pediatric Gastroenterology and Nutrition: September 2005 - Volume 41 - Issue 3 - p 273-285
doi: 10.1097/01.mpg.0000178439.64675.8d
Special Feature: Special Report

For all Participants of the Cystic Fibrosis Foundation Workshop

Received July 1, 2005; accepted July 7, 2005.

This workshop was sponsored by the Cystic Fibrosis Foundation.

Individual authors were supported by National Institutes of Health grants DK048816 (LLC), DK 56791 (RCD), HD 033060 and DK 053100 (MEL), R42 DK48537 and R44 DK48190 (MJ) and Sheffield Children's Hospital Charity, Special Trustees Sheffield Hospitals Charitable Trust, Cystic Fibrosis Trust (CJT), and Solvay Pharmaceuticals (RB).

JS is compensated and has an equity position in SmartPill™. MJ is the president and part owner of BioChemAnalysis Corporation.

Until the mid 20th century, children with cystic fibrosis (CF) died at a young age from a combination of malnutrition and suppurative lung disease. In the past three to four decades, coincident with new treatments for pulmonary complications and the use of high-calorie diets without restrictions on fat intake, there has been marked and progressive improvement in lung function, nutritional status and survival. However, treatment for pancreatic insufficiency, which affects most individuals with CF, remains largely unchanged, and there have been few advances in our understanding and management of gastrointestinal problems in this population.

Pancreatic Enzyme Replacement Therapy (PERT), principally using enzyme extracts of porcine origin, has been the mainstay for treating maldigestion resulting from pancreatic insufficiency (PI) for over a century. Powdered PERT was instituted before the 1931 creation of the United States Food and Drug Administration (FDA) and the subsequent Federal Food, Drug and Cosmetic Act of 1938 that required proof of safety and efficacy before approval of drugs for marketing. Enteric-coated microcapsules, the most commonly used PERT, have never been FDA approved. After the epidemic of fibrosing colonopathy in the early 1990s, a condition that was clearly related to high doses of exogenous pancreatic enzymes (1), the FDA initiated a process of re-evaluating the safety and efficacy of PERT. In 2004, the FDA released a draft of a new Guidance Document for public comment (2) that stated that within the next 4 years, all manufacturers of current enzyme products will need to submit a New Drug Application to the FDA with proof of safety in manufacturing, stability and efficacy.

The coefficient of fat absorption (CFA) is the traditional method of testing the clinical efficacy of PERT. CF clinicians have long recognized that there is a very wide range of CFA in patients with CF. Less well known is that there is marked variability in the actual amount of enzyme in each capsule. Capsules are overfilled so that the stated dosage reflects the least amount of enzyme activity present after a six-month shelf life. There is little correlation between CFA and enzyme dose when subjects are receiving a dose of PERT considered optimal by the caregiver and/or patient (3). Furthermore, a recent cross-sectional study showed no correlation between enzyme dose and growth or self-reported symptoms (4). Consequently, the common belief that PERT dose can be titrated to correct malabsorption and/or maldigestion using patient-reported relief of abdominal symptoms or stool bulk and consistency appears to be questionable. PERT may not completely correct PI even if given in adequate doses because the release and onset of activity is not synchronous with the presence of food in the proximal intestine, the site for optimal absorption. Furthermore, as discussed below, it is becoming evident that poorly understood nonpancreatic intestinal and/or hepatobiliary factors likely contribute to incomplete and variable nutrient assimilation in patients with CF. However, we lack tools to distinguish PERT-related factors that might contribute to treatment failure from other confounding factors.

Ironically, as mouse models of CF were developed in the late 20th century, these animals did not develop lung or severe pancreatic manifestations. However, the mice have characteristic CF-like bowel obstruction and are proving to be excellent models for study of nonpancreatic gastrointestinal manifestations of CF.

The combination of new ways to study gastrointestinal disease in CF that further our understanding of the pathophysiology of CF and the need to evaluate novel methods to study pancreatic and nonpancreatic causes of maldigestion and malabsorption led the CF Foundation to sponsor a workshop, which was held in Baltimore in May 2005. The goals of this workshop were to evaluate what is known about gastrointestinal issues leading to malabsorption, to consider other factors that may contribute to malabsorption and to identify ways to improve care of patients with CF. We set an agenda for further clinical and laboratory-based investigations that would help to elucidate the spectrum of causes of maldigestion and malabsorption. The workshop participants included clinicians and investigators with a variety of interests, members of the US and European regulatory agencies and individuals from industry who were involved in producing traditional PERT or developing novel PERT or diagnostic tests.

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Classic CF reflects two loss-of-function CFTR mutations whereas patients with nonclassic CF carry at least one copy of a mutant gene that confers partial function of the CFTR protein (5). Patients with “classic” and “nonclassic” CF exhibit wide heterogeneity in the severity of lung disease. However, in statistical terms, the overall rate of progression of lung disease among the nonclassic CF patients is slower and median survival is almost twice that of patients with “classic” CF.

Among the various gastrointestinal organs affected by CF disease, the exocrine pancreas shows the strongest association between genotype and phenotype. Loss-of-function mutations on both alleles almost always confer pancreatic insufficiency (PI). In most cases, considerable obstructive destruction of the pancreas arises in utero and functional loss of the exocrine pancreas develops at birth or in early infancy. These patients need PERT with meals and snacks. In patients with the pancreatic sufficient phenotype the presence of at least one mutation with some residual CFTR function protects the pancreas from complete destruction. However, the functional capacity of the exocrine pancreas in the patients who are pancreatic sufficient varies widely, from values just above the threshold for developing PI to values that are within the reference range for healthy controls (6). Approximately 20% of pancreatic sufficient patients are at risk of developing symptoms of recurrent, acute pancreatitis or chronic pancreatitis.

In contrast to the exocrine pancreas, expression of disease in other gastrointestinal organs is less clearly associated with CFTR genotype. However, patients with classic CF are more vulnerable than their nonclassic counterparts to meconium ileus (MI), distal intestinal obstruction syndrome (DIOS) and clinically significant liver disease (CFLD). As disease penetrance is incomplete, disease expression seems to depend on two loss-of-function CFTR mutations as well as the influence of modifier genes and/or environmental factors.

Approximately 25% of neonates with classic CF develop MI. A higher incidence of MI is seen among subsequent born siblings with CF within families in which the first-born child had MI. This suggests a genetic contribution to MI. Linkage and association analysis of different murine strains of CF led to the identification of two candidate regions for modifier genes of MI. The strongest linkage (CFM1) was detected on proximal murine chromosome 7. Subsequently, a modifier locus for CFM1 was detected in the syntenic region of human chromosome 19q13 using DNA from CF siblings who were concordant or discordant for MI. As CFM1 showed no association with pulmonary function, a gene in the CFM1 region probably acts solely within the small intestine and together with two severe CFTR alleles increases the likelihood of MI (7).

Most patients with CF develop mild liver abnormalities including slightly elevated liver biochemical tests, hepatosteatosis and focal areas of portal tract disease (biliary plugging with eosinophilic material, bile duct proliferation and cholangitis). Only a small proportion of CF patients, most of whom have classic CF, develop CFLD with multilobular cirrhosis leading to portal hypertension and hypersplenism. The median age at diagnosis of CFLD is 9-10 years and most cases of CFLD are diagnosed by midadolescence. Because the ability of the liver to synthesize proteins, metabolize toxins and secrete bile tends to remain intact for many years and even decades, most complications of CFLD are attributable to the consequences of portal hypertension. A number of putative modifier genes of CFLD have been reported in recent years. However, in a recent study of a larger number of patients with severe CFLD, only two candidate genes (the Z allele for alpha-1-antitrypsin deficiency and polymorphisms of TGFβ1 with increased expression) were shown to be statistically associated with CFLD (8).

Taken together, the CFTR genotype, modifier genes and environmental factors appear to explain the variability in the severity of the CF phenotype in different organs (Fig. 1). However, the relative contributions of these factors vary from organ to organ. In the case of the exocrine pancreas, CFTR genotype seems to dictate the severity of disease. In contrast, expression of disease in other organs, such as the small intestine and the liver, requires the influence of severe CFTR genotypes on both alleles as well as modifier gene and/or potential environmental influences.

FIG. 1

FIG. 1

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CF has not been reported as a spontaneous disease entity in any species other than humans. This has led to the development of 13 genetically-manipulated mouse models including seven with gene-targeted disruption of the murine CFTR (knockouts), three mutant models expressing the ΔF508 mutation and mouse models with the G551D, R117H or G480C mutations (Table 1). The major manifestation of CF in the knockout mouse is obstruction of the distal intestine, which commonly results in bowel perforation, peritonitis and death. Intraluminal obstruction occurs at two stages; the immediate perinatal period (recapitulating MI) and after weaning to solid chow (recapitulating DIOS). Bowel obstruction in mouse models with severe disease occurs with high frequency (>90%), significantly exceeding the incidence of MI or DIOS in humans. The incidence of obstructive bowel disease in CF mouse models is closely associated with abnormalities in electrolyte transport across the epithelium, which in turn leads to goblet cell hyperplasia and dehydration of secreted mucus and debris in the crypts. Obstructing impactions can be prevented by several dietary methods such as including osmotic laxatives in the drinking water (polyethylene glycol-based), use of a liquid diet (Peptamen® or Liquidiet®) and, more recently, by providing a calorie-dense diet (9-11).



In both mouse and human, CFTR is the predominant apical anion channel in the intestinal epithelium (9,10). Loss of CFTR greatly diminishes basal and stimulated anion secretion, including the loss of transepithelial bicarbonate secretion in the upper small intestine (12-14). Furthermore, the absence of CFTR function in the villus epithelium of the proximal bowel results in deficient cyclic adenosine monophosphatase regulation of intestinal Na+ absorption, which further exacerbates luminal dehydration (15). The severity of gastrointestinal obstructive disease in the CF mice varies with the genotype (targeting strategy) and with the genetic background of the mouse strain. Knockout or ΔF508 CFTR mouse models generated by replacement of a portion of the endogenous gene by vector sequences typically have very low levels of CFTR gene transcription, significantly impaired anion secretion and severe intestinal disease (e.g., Cftrtm1Unc or Cftrtm1Cam models). In contrast, mouse models generated by insertion or “hit and run” gene-targeting (no replacement of the endogenous CFTR sequence) have residual expression of ∼10% wild-type CFTR by alternate splicing in the knockout (Cftrtm1Hgu mice) or sufficiently high levels of ΔF508 CFTR that allow “escape” of the mutant protein from the processing defect (e.g., Cftrtm1Eur mice). These models typically have significant anion secretion and mild intestinal disease (9).

The severity of intestinal disease also varies with the genetic background of the CF mice. The importance of background was elegantly demonstrated by Rozmahel and coworkers, who showed that only a small percentage of CFTR knockout mice with mixed background had long-term survival in the absence of palliative treatment (16). When the responsible mutation was inbred to a BALB/cJ background, the percentage of long-term surviving mice increased and this was subsequently correlated with an increased capability for non-CFTR-mediated anion secretion via alternate Cl conductance pathways (17). Despite the varying influences of genetic background, diet and targeting strategy, the accumulated data on CF mouse models show two important correlations. First, the survival of CF mice is a positive linear function of the magnitude of residual anion secretion. Second, the degree of growth retardation of the CF mice correlates in a linear fashion with the magnitude of stimulated anion secretion.

Because the expression of CF disease in the airways and pancreas of CF mice remains subclinical, these animal models can be used to isolate deficiencies in mucosal digestion and intestine absorption. Although significant hepatic disease is not evident in young CF mice, one study of long-lived CF mice found progressive hepatobiliary changes that mimic the disease of adult CF patients (18).

CF mouse models display several other intestinal features in common with CF patients that may be of value for evaluating factors that contribute to mucosal malabsorption. First, CF mice with severe disease phenotypes have acidic, weakly sulfated surface intestinal mucus that may alter its functional properties and affect nutrient diffusion or negatively influence the juxtamucosal environment. Second, consistent with recent studies of human intestine, CF mice have low-grade intestinal inflammation that might be secondary to deficiencies in host defense or microbial intestinal interactions (19).

Postsecretory Paneth cell granules, which are rich in α-defensins (cryptdins) and other antimicrobial peptides, do not undergo normal dissolution in the intestinal crypts. This increases the propensity for bacterial colonization of the small intestine (20). CFTR acts as a HCO3 unloading mechanism for the epithelial cell. Abnormalities of intracellular pH regulation in CFTR-null intestinal epithelia have been associated with increased enterocyte proliferation in the upper intestine that may alter nutrient transporter expression along the crypt-villus axis (21).

The experimental conditions for evaluating nutrient maldigestion and malabsorption in CF mouse models must be carefully designed with regard to background strain and diet. For example, early investigations of CFTR knockout mice with a mixed background and consuming a liquid diet (Peptamen®) were shown to have an essential fatty acid imbalance (22). However, an essential fatty acid deficiency was not detected in congenic CFTR knockout mice consuming either a conventional diet or Peptamen® (23). In a follow-up to the latter study, it was shown that CFTR knockout mice have steatorrhea and reduced mucosal lipolysis that was indirectly related to a deficiency in epithelial bicarbonate secretion (11). Earlier investigations of Na+-coupled glucose transport in CFTR knockout mice with varying backgrounds and diets did not find differences from wild-type littermates (10), whereas a more recent study of ΔF508 CFTR mice with congenic background report reduced glucose absorption (24). The latter study contrasts with previous investigations of human CF intestine that show either increased or unchanged glucose absorption. However, these studies report large differences in the absolute rates of Na+-coupled glucose transport between the two species. Although amino acid and peptide absorption in CF mice has received little attention, neonatal CFTR knockout mice have been reported to show decreased Na+-coupled alanine transport (10). Thus there is a need to evaluate the array of amino acid transport systems (currently 17 systems) and H+-coupled peptide transporters. Accessory proteins that support the transport process also need to be considered. The apical membrane Na+/H+ exchanger isoform NHE3 is activated during Na+-coupled glucose and H+-coupled dipeptide absorption, ostensibly to maintain intracellular pH (25,26). A recent study has shown that intestinal NHE3 activity is decreased in CF mice with severe disease phenotypes (27). Whether this contributes to carbohydrate and peptide malabsorption remains unknown.

Recommendations for investigations of mucosal maldigestion and malabsorption using CF mouse models can be offered. First, to accentuate the role of CFTR in nutrient absorption, mice with severe intestinal disease are recommended (CFTR knockout, ΔF508 CFTR by replacement gene-targeting strategies). Second, it is necessary to use CF mice bred in congenic backgrounds to avoid variability of disease phenotype. Most studies using a C57BL6/J background report a severe intestinal disease phenotype, whereas a BALB/cJ background might accentuate the role of the alternate Cl conductance. Third, a strong case can be made against the use of liquid diets, which do not provide the beneficial effects of dietary bulk or food antigens. Some liquid diets, such as Peptamen® are not appropriate for rodent metabolism because a large volume of fluid (∼75% of body wt.) must be consumed in a 24-hour cycle and amino acid containing diets may not adequately stimulate CCK release. Special conditions are also required to prevent malocclusion of continuously growing rodent incisors. In contrast to liquid diets, CF mice on calorie dense diets without palliative treatment may have confounding pathology related to impending impaction. Thus, optimal studies of mouse digestion and absorption may require the use of osmotic agents, such as polyethylene glycol containing solutions, which offset luminal dehydration and allow conventional mouse diets to be used. Only age-matched siblings [CFTR (+/+) or (+/−)] should be used as controls because age-related changes in intestinal transport properties can be detected even in congenic strains. Finally, it should be recognized that all animal models have intrinsic limitations resulting from differences between strains and species.

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Although mouse models can help us understand basic aspects of gastrointestinal pathophysiology in CF, clinicians are faced with a number of pressing, poorly understood clinical problems that adversely affect patients and limit our ability to conduct meaningful clinical studies. One such challenge has been our inability to dissect out the factors that may contribute to the prevalence of gastrointestinal complaints and the wide range of severity of nutrient maldigestion and malabsorption, as well as factors that perturb the effectiveness of current PERT. A number of factors might contribute to these difficulties.

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Gastric Emptying

Gastric emptying of food and enzymes may not be synchronous. Although a variety of methods for assessing gastric emptying have been described, the most widely used is serial scanning of radiolabeled solid and/or liquid meals. The patterns of gastric motility and the rates of gastric emptying of viscous nonnutrient meals (e.g., inert cellulose) differ from nutrient meals (e.g., casein, glucose, or oleic acid). The rate of delivery of inert liquids into the duodenum is a constant fraction of the volume of liquid within the stomach. For example, 250 mL of saline will empty twice as fast as 125 mL. Nutrients within the stomach slow gastric emptying because of feedback control from the small intestine. The rate of emptying of liquid meals is directly related to the caloric content of the meal within a given range. Digestible solids must be ground to 1-2 mm particles before emptying occurs. Smaller particles are emptied first. The particle size of most PERT is smaller than 2 mm so, in theory, they would be expected to empty mixed with a meal. Powdered enzymes would be expected to empty faster with the liquid phase of the meal.

There have been eight studies of gastric emptying in CF (28-35). No two studies used the same test meal, and five different test methods were used. No study evaluated both solid and liquid phase emptying. The results are difficult to interpret because three studies showed rapid emptying, four showed normal emptying and one showed delayed emptying. Most studies evaluated a small number of individuals and the reference measures were often incompletely defined. The degree of intra-patient variability in gastric emptying was not evaluated. This is important because the degree of intra- and inter-patient variability in gastric emptying is considerable even in healthy subjects and is influenced by multiple factors including physical or emotional stress, composition and volume of gastric contents, vagal reflexes, propulsive motility of the antral pump, compliance of the gastric reservoir, feedback signals from the duodenum and the ileal brake phenomenon. One study of patients with CF evaluated simultaneous emptying of food and PERT using a double-labeled radionuclide technique. There was wide inter-patient variability in the rate of gastric emptying and intestinal transit of food. Furthermore there was considerable asynchrony between emptying of PERT and nutrients, although enzyme pellets generally emptied faster than food from the stomach (36). Another study found a significant negative correlation between the rate of gastric emptying and lipolysis as measured by 13C-mixed triglyceride breath testing (34). A third study showed improved recovery of 13CO2 from 13C-mixed triglyceride when enzymes were taken during or after a meal rather than before the meal. However, roughly half of subjects did not normalize recovery of 13CO2 with any dosing regimen (37).

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Intestinal Acidification

Low intestinal pH and gastric pepsin may inhibit both endogenous and exogenous pancreatic enzyme activity. Duodenal pH is regulated by bicarbonate-rich pancreatic secretions and intestinal bicarbonate secretion. CFTR is known to play an important role in both routes of bicarbonate secretion. Duodenal bicarbonate secretion by the epithelium involves two pathways: electroneutral secretion via a CFTR-assisted Cl/ HCO3 exchange process and an electrogenic secretion of HCO3 via a CFTR conductance pathway (12). Exposure of the duodenal mucosa to an acidic pH triggers HCO3 secretion via pathways that include prostaglandin release and neural activity. Basal HCO3 secretion is reduced in the CF duodenal mucosa, and, in contrast to normal controls, cyclic adenosine monophosphatase-stimulated HCO3 secretion is absent (13). Although Cl/HCO3 exchange provides basal HCO3 secretion in the CF intestine, the magnitude of secretion is lessened by simultaneous activity of a Na+/H+ exchanger. During cyclic adenosine monophosphatase stimulation of the CF duodenum, a small net increase in base secretion can be measured as a result of cyclic adenosine monophosphatase inhibition of Na+/H+ exchange activity rather than increased HCO3 secretion (14).

Most commercially available PERT are coated with an acid-resistant film to prevent the pancreatic enzymes from being denatured within the acidic environment of the stomach. Reduced pancreatic and duodenal bicarbonate secretion may fail to neutralize gastric acid and thereby prevent or delay dissolution of the enteric coating until the microspheres have passed the major absorptive surface area in the duodenum and jejunum. Even if the coating dissolves, the activity of most pancreatic enzymes, particularly pancreatic lipase/colipase, is greatly impaired in an acidic intraluminal environment. In theory, duodenal pH can be made more alkaline by inhibiting gastric acid secretion with the use of H2-receptor antagonists or proton pump inhibitors (PPI) (38). PPIs can decrease malabsorption in patients with CF (39). Treatment with a PPI partially corrected fat malabsorption that was present in CF mice lacking a lipolytic defect (11). Abnormal intestinal acidification may contribute to fat malabsorption in CF irrespective of its effect on pancreatic enzyme activity.

In vivo monitoring of intestinal pH in an ambulatory clinical setting may be available in the near future. The SmartPill™ gastrointestinal monitoring system (SmartPill, Buffalo, NY) uses an ingestible radio telemetry capsule to characterize pressure and pH of the entire gastrointestinal tract. The system includes a small radio frequency receiver, docking station and data processing software along with the 1 in. × 1.5 in. ingestible capsule. The SmartPill™ contains microelectronic pH, pressure and temperature sensors and an internal power supply, transmitter and microprocessor. The device is capable of recording gastrointestinal pressure, pH and temperature for more than 3 days. Once approved by the Food and Drug Administration, this device may make it easier to define intestinal acidification profiles in patients with CF.

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Dietary fats are essential for health. They are an important source of calories, supply precursors for cellular membranes, prostaglandins, thromboxanes and leukotrienes, present a vehicle for fat-soluble vitamins and improve the palatability of foods. Triglycerides comprise approximately 95% of the fats in a typical Western diet. Phospholipids and cholesterol account for the majority of the remaining fats. In addition to the diet, intraluminal fats come from biliary lipids and mixed membrane lipids from desquamated intestinal cells and dead bacteria. The majority of ingested lipids are absorbed by intestinal enterocytes and less than 5% are normally excreted in the stool.

Before enterocytes can absorb fats, the lipids must be digested into their component parts and made soluble. Triglycerides are digested into fatty acids and monoacylglycerols whereas phospholipids are digested into fatty acids and lysophosphatides. Digestion proceeds at the surface of multilamellar emulsion particles through the action of lipases. First, gastric lipase releases approximately 15% of the fatty acids during digestion in the stomach. Then pancreatic lipase, in conjunction with colipase, preferentially cleaves at the sn-1 and sn-3 positions, thereby completing the process in the upper small intestine. As digestion proceeds, fatty acids, monoacylglycerides, lysophosphatides and cholesterol form unilamellar vesicles and intestinal mixed micelles by mixing with bile salts. Most fat absorption occurs from the micelles, which increase aqueous solubility of lipids up to 1,000,000-fold compared with monomer concentrations (39-41).

The increased solubility enhances intestinal absorption of the digested lipids by facilitating diffusion across the unstirred water layer that is contiguous with the intestinal membranes. The unstirred water layer provides an aqueous barrier to the diffusion of monomeric fatty acids but is readily traversed by mixed micelles. Once the mixed micelles approach the intestinal membrane they enter an acidic microclimate where the fatty acids are protonated, destabilizing the micelle and facilitating translocation of the fatty acids across the plasma membrane of the enterocyte. It is still unclear whether entry into the intestinal cells occurs through passive diffusion or carrier-mediated transport. Fatty acid binding proteins inside the cell may act as a sink to remove the fatty acids from the plasma membrane and increase the diffusion gradient and thereby transport across the membrane. Once inside the cells, acyl synthetases re-esterify the fatty acids into triglycerides and phospholipids before being packaged into lipoprotein particles for distribution throughout the body. Defects in CFTR can theoretically affect the postlipolytic process. For example, there is evidence that long chain monomeric fats and phospholipids are malabsorbed in CF (42,43). The clinical consequences of phospholipid malabsorption in CF are unknown.

Animal models suggest a role for deranged bile salt pharmacokinetics in CF. Fat absorption in two murine CF models was determined by measuring fat excretion in stool. Lipolysis and postlipolytic long chain fatty acid uptake was assessed by comparing uptake of 3H-triolein-derived lipid and 14C-oleate (11). Biliary function was investigated by assessing bile salt secretion rate and composition and kinetics of the enterohepatic bile salt circulation using a stable isotope dilution method. Both CFTR null mice and homozygous ΔF508 mice had increased fecal bile salt losses and increased bile salt secretion rates compared with controls. Uptake of 14C-labeled dietary lipid in intestinal mucosa and appearance in plasma was significantly reduced, indicating that postlipolytic events were impaired. Only the CFTR null mice had steatorrhea, although there was no evidence of reduced pancreatic secretion. Of note, this maldigestion could be partially restored by suppression of gastric acid. Although both the CFTR null and homozygous ΔF508 mice had abnormal bile salt pharmacokinetics, the fact that the homozygous ΔF508 mice did not have steatorrhea suggests that the contribution of abnormal fat solubilization by bile salts to fat malabsorption is minimal. Likewise, humans with CF have been found to have high fecal bile acid excretion, but this is poorly correlated with fecal fat losses (44). It is possible that the excessive fecal bile acid losses seen in both humans and CF mice are a clue to the abnormal terminal ileal function that must be a part of both MI and DIOS, which bears further exploration.

Epithelial damage and/or dysfunction may contribute to fat malabsorption in humans, similar to findings in CF mice. At the mucosal level, it has been shown that lactase and alkaline phosphatase activity are reduced in children with CF (45), whereas duodenal sucrase-isomaltase activity is apparently unaltered. The 13C sucrose breath test (46) has been proposed as a potential method of assessing the functional health or intactness of the small intestinal mucosa.

Taken together, these studies suggest that loss of CFTR function influences a number of postlipolytic factors that contribute to fat absorption, including micellar solubilization of lipids and epithelial transport of both bile salts and nutrients.

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Bacterial Overgrowth

The relationships among intestinal bacteria and the body are complex and are just beginning to be explored in depth. As many as 500 different bacterial species reside in the normal human gut. In health, the small intestine is relatively sterile and bacteria are largely confined to the terminal ileum and especially to the cecum and colon (104 organisms/mL in the proximal small intestine versus 1012 organisms/mL in the colon). In some circumstances the proximal small intestine is inappropriately colonized with bacteria. Small intestinal bacterial overgrowth (SIBO) occurs with the failure of one or more mechanisms for maintaining relative sterility of the small intestine.

SIBO can cause inflammation and damage to intestinal mucosa and may result in diarrhea, steatorrhea, macrocytic anemia and weight loss (47). Weight loss may be attributable to bacterial competition for ingested nutrients as well as intestinal inflammation from enterotoxic metabolites. Inflammation may cause maldigestion and malabsorption of fat, carbohydrate and protein. The bacteria also deconjugate bile acids, reducing their ability to emulsify fat. Taken together, these factors may contribute to the severity of fat malabsorption, thereby worsening the loss of calorie dense nutrients and fat-soluble vitamins.

Although the significance of SIBO in CF has received limited attention to date, tests of low sensitivity suggest that SIBO is present in 30% to 50% of those studied (44,48,49). Theoretically, CF disease-related risk factors and some therapeutic interventions may predispose patients to SIBO:

  1. Gastric acid which normally sterilizes ingested material (including swallowed airway mucus) is often suppressed therapeutically to protect lipase activity;
  2. Regular flushing of the intestine may be impaired by reduced pancreatic, biliary and crypt enterocyte fluid secretions and slowed intestinal transit;
  3. Abnormal accumulation of surface mucus in the poorly-hydrated and acidic lumen of the CF intestine may allow bacterial adhesion and proliferation; and
  4. Altered biophysical and biochemical properties of intestinal mucins may alter their protective functions making the intestinal mucosa more susceptible to the harmful effects of bacteria.

In clinical practice, there are direct and indirect methods of testing for SIBO (50,51). Purportedly, the standard procedure is direct aspiration of intestinal contents for culture, but the procedure is limited by its invasive nature and the cost and labor intensity of establishing cultures. Furthermore, in patients with CF, bacteria may be adherent to the mucosa, potentially covered by a thickened mucus layer and thus inaccessible by simple aspiration. To avoid these issues, breath tests have been developed to diagnose SIBO. Although the 14CO2-xylose breath test is considered to be the most accurate, it is rarely used in clinical practice and is contraindicated in children. 13C-xylose could be a safe alternative, but it is not commercially available. The lactulose breath hydrogen test is most commonly used in clinical practice (52), but this method results in poor sensitivity and specificity. Other potential limitations include test confounders such as intestinal inflammation and misinterpretation of labeled-carbon breath in CF patients with CO2 retention (53). End-tidal breath may be difficult to sample in infants with tachypnea and in patients with severe obstructive lung disease.

Despite the lack of an easy, reproducible clinical test for SIBO and limited information in CF specifically, there is direct and indirect experimental evidence to reinforce the concept that SIBO may play a significant role in malabsorption in CF. The prominent histologic feature of the CF mouse small intestine is mucus accumulation, which occludes the crypts and coats the villus surfaces. Mucus obstruction of the crypts is believed to interfere with innate defense mechanisms of the Paneth cells that reside at the base of the crypts and secrete a variety of antibacterial products (20,54). The CF mouse small intestine also shows evidence of mild innate inflammation, characterized by increased mucosal infiltration of mast cells and neutrophils (19).

A greater than 40-fold increase in luminal bacteria in the CF mouse small intestine has been found, and bacteria were observed to colonize the mucus along the villus surfaces (54). Based on these observations, one could speculate that SIBO of the CF mouse small intestine is similar to that reported in humans. Analysis of bacterial 16S ribosomal genes has been used to classify the intraluminal bacteria of the wild-type and CF mice. There was reduced species diversity in the CF intestine, with more than 90% of the microbes belonging to the Enterobacteriacae family. Also, approximately 6% of the bacteria in CF mice were Clostridium perfringens, which was not detected in the wild-type animals. The presence of C. perfringens in the CF gut may be significant, as it produces enzymes capable of deconjugating bile salts.

To explore the potential link between SIBO, small intestinal inflammation and failure to thrive in CF mice, Norkina et al (54) conducted a treatment trial of broad-spectrum antibiotics given orally. The treated CF mice had less inflammation of the small intestine, and gained significantly more weight than their untreated CF littermates, being only about 10% smaller than wild-type mice instead of the 30% difference in untreated CF mice. These data suggest that management of the intestinal microbial load and/or species composition may improve nutrient absorption and nutritional status of CF patients.

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Other Pathogens

Giardia lamblia, a common flagellate protozoan, can cause steatorrhea and is associated with protracted or intermittent passage of foul-smelling stools, flatulence, abdominal distention and anorexia. Older data suggest that giardiasis may be more common in individuals with CF than in other household members (55). Stool specimens can be evaluated for Giardia by direct examination of stool or by detection of Giardia antigens by enzyme immunoassay, which is more sensitive than microscopy of a single specimen.

Clostridium difficile can elaborate a toxin that causes severe enterocolitis. Despite the fact that patients with CF are frequently and chronically treated with broad-spectrum antibiotics and that there is a somewhat increased rate of recovery of toxigenic C. difficile in their stool, symptoms are uncommon (56,57). However, there have been several case reports of acute toxic megacolon as the presenting symptom, which may be life threatening. It is unclear whether subclinical infections contribute to malabsorption in patients with CF by causing direct mucosal damage or whether patients with CF develop acquired tolerance to C. difficile toxin.

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Until the middle of the last century CF and celiac disease (CD) were not distinguished from each other. With the recognition of CF as a separate disease and development of better techniques for establishing a diagnosis of these conditions, reports of the two diseases occurring in the same individual began to appear in the literature. Lloyd-Still provided evidence in support of an increased risk of CD in patients with CF in 1994 (58). Despite some methodological flaws, this epidemiologic study appeared to demonstrate a fivefold increase in the prevalence of CD in the CF population. CD is a multigenic disease that requires human leukocyte antigen DQ2 or DQ8 expression. Although exposure to gluten is essential, not all genetically susceptible patients develop CD. Thus, there is a requirement for additional genetic or environmental factors. On this basis it is postulated that CF might be one such second “hit” (59).

With the advent of sensitive serological screening methods it has become evident that CD is much more common in the general population than previously believed. In fact, patients with CD who present with symptoms represent a tip of the iceberg. Underneath that “tip” there are considerably more asymptomatic patients who have positive serology as well as histologic evidence of CD; below that are many patients with “latent” disease, who have positive serology but no mucosal damage. The frequency of CD is now thought to be as high as 1/133 on the general population (60,61). Thus there could be many undiagnosed CD patients among the CF population. Now that highly sensitive and specific serologic tests are available, systematic screening to determine the true prevalence of CD in CF populations seems quite feasible (62).

Crohn disease is another multigenic disease that is said to occur with increased frequency in CF. Specifically, the epidemiologic study of Lloyd-Still (58) suggests a 12.5-fold increase in the prevalence of Crohn disease in patients with CF than in the general population.

Genetic mapping studies have isolated an important susceptibility gene (NOD2) as well as a number of Crohn susceptibility loci on chromosomes 1,5,6,12,14,16 and 19 (63). As with CD, it has been postulated that Crohn disease may become manifest in a genetically susceptible individual as a result of one or more second “hits”. Aberrant immunologic interactions with gut microflora, pathogenic bacteria or bacterial antigens are all hypothesized as examples of second hits. It is possible that CF disease increases susceptibility to Crohn disease through an unknown CF-related intestinal mechanism. Possibilities include increased intestinal permeability and inflammation, alterations in the intraluminal environment resulting from maldigestion, the presence of bacteria in the small intestine or changes in gut microflora.

Unlike CD, serological assays for Crohn disease and ulcerative colitis (ASCA and pANCA) (64) are not sufficiently sensitive or specific for screening purposes to confirm or refute Lloyd-Still's observations more extensively. Nevertheless, based on current information, it is important to retain a high index of suspicion and consider the possibility of Crohn disease in CF patients who fail to respond to usual CF therapy.

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Distal intestinal obstruction syndrome (DIOS) is an incomplete or rarely complete intestinal obstruction in the ileo-cecal region. It is a complication unique to CF that is generally seen in a subset of older children and adults. Although the etiology is unknown, it appears to be limited to patients with the PI phenotype. However, observations in CF knock-out mice indicate that loss of pancreatic function per se does not exclusively correlate with bowel obstruction. Instead, the genetic background of mouse strains greatly influences the frequency and severity of DIOS. Thus, non-CFTR modifier genes in the presence of severe CFTR mutations on both alleles are likely to play a role in its pathogenesis in humans. By inference, attempts to control the symptoms of DIOS by increasing the dose of PERT may not be of therapeutic value. Because identical food products from different vendors showed dramatically different effects on bowel obstruction in CF mice, as yet undefined nutritional factors may also contribute to the development of DIOS in humans.

By self-report, constipation is common in patients with CF and is strongly associated with self-report of gassiness (4). Although the true incidence of constipation in CF is unknown, it is common in the general population and is unlikely to be less frequent in CF. Complaints of gassiness may respond to treatment of constipation rather than an increase in PERT. Functional bowel complaints are also common, occurring in 20% to 30% of the general population. We do not yet have methods to distinguish between abdominal pain related to visceral hypersensitivity (irritable bowel syndrome) and the various CF causes of abdominal pain. Irritable bowel syndrome should be considered in the differential diagnosis of a patient with abdominal pain, bloating and diarrhea or constipation, especially if other family members have visceral hypersensitivity.

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The CFA is the standard method for evaluating fat absorption. It is not ideal, as it cannot distinguish between pancreatic maldigestion and other causes of malabsorption, has poor test-retest reliability and is odious to perform. For greatest accuracy the stool collected should represent stool produced during the period of ingesting a measured high fat diet (100 g fat/day or 60 g fat/m2). Ideally the patient should be on the correct diet for several days before initiating the stool collection, particularly when testing PERT therapy. Oral dye markers taken at the beginning and end of the period of high fat intake demarcate the stool to be collected to determine fat losses. Traditional markers such as charcoal and carmine red can be difficult to detect in stool. Carmine blue (FD&C Blue #2) is readily detectable and may improve test accuracy (65). Incomplete collection of stool (contributing fewer grams of excreted fat to the CFA calculation), a low-fat diet or an incomplete diet history (contributing to fewer grams of fat ingested to the CFA calculation) could lead to incorrect estimates of fat malabsorption. Before enrolling subjects for studies of malabsorption, subjects should have objective evidence of PI by assessing CFA off enzymes or performing fecal elastase. Some studies of PERT efficacy in CF have inadvertently included pancreatic sufficient subjects; this artificially increases the group mean CFA (66,67).

Alternatives to CFA are urgently needed for clinical care and research. Potential alternatives to measure fat absorption and to distinguish maldigestion from malabsorption include measuring absorption of fats or their digestive products in the blood, measuring metabolites of absorption in breath or measuring malabsorbed fecal fat using a nonabsorbable marker in a single stool sample. The last method is a paradigm shift away from lengthy stool collections, similar to the trend away from using 24-hour urine collections by normalizing spot collections against a dilution factor.

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Blood Absorption Tests

Measurement of products of absorption in blood after a fatty meal is not a new concept (68), but it has recently been restudied in a methodical way (69). The Malabsorption Blood Test uses two naturally occurring lipid substrates: a fatty acid, pentadecanoic acid (C-15), and a triglyceride, triheptadecanoic acid (C-17). A study meal composed of chocolate Scandishake® mixed with soy milk, microlipids and a fixed and equimolar dose of the two lipids (5.0 g of pentadecanoic acid and 5.5 g of triheptadecanoic acid) is employed. The test meal contains 550 calories and 32 g of fat with 52% of calories derived from fat. Because pentadecanoic acid is a free fatty acid it is absorbed independently of pancreatic digestion, whereas triheptadecanoic acid is a triglyceride and thus requires hydrolysis by pancreatic lipase/colipase before absorption. Mathematical modeling techniques have been used to determine the difference in blood levels of the two substrates, which in turn define the degree of fat malabsorption attributable to PI in patients with CF (and potentially in other diseases with pancreatic or nonpancreatic causes of malabsorption and/or maldigestion). Currently, test refinements include assessing test-retest reliability and correlations with CFA, among other issues. This test has the potential to distinguish pancreatic from nonpancreatic causes of fat malabsorption.

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Breath Tests

The noninvasive nature and relative simplicity of breath tests, using breath hydrogen or 13CO2 excretion offer a potential vehicle to measure a variety of dynamic processes involving coordinated delivery and processing of nutrients to the small and large intestine (70). For example, the impact of aberrant and uncoordinated emptying of a meal and pancreatic enzyme microspheres can be assessed using a 13C octanoate breath test (34,71). The effect of altered gastric emptying on the efficiency of lipase activity and fat absorption can be measured using a 13C mixed triglyceride breath test or 13C triolein breath test (72,73). However, 13CO2 recovery after ingestion of mixed triglyceride had poor correlation with dietary fat absorption in a small number of subjects with CF (42). A study in rats fed high-fat chow with varying amounts of the lipase inhibitor orlistat suggested that the 13C mixed triglyceride test could potentially replace fat balance studies for comparing fat absorption between groups (74). However, the large interindividual variation under conditions of mild fat malabsorption may limit its use for diagnostic purposes in individuals with less severe PI.

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Spot Stool Samples

13C-labeled Triglyceride Plus Dysprosium

An intriguing concept for determining fecal fat absorption is based on co-administration of an appropriate13C-labeled triglyceride (TG*), the nonabsorbable gastrointestinal marker dysprosium chloride (Dy) (75) and a visual stool marker with a meal. Assuming intestinal transit of the three substances is the same, the amount of 13C-excess and Dy can be measured from two small aliquots within the stool colored by the visual dye marker. The ratio of fecal TG* and Dy permits quantitative evaluation of fecal TG* excretion.

For the TG*-Dy Method to quantitatively reflect CFA a number of criteria must be met: a) Dy from dysprosium chloride must have negligible gastrointestinal absorption in CF patients, b) intestinal transit kinetics of Dy, TG* and the stool colorant must be identical and indistinguishable for the wide range of dietary fat losses that is normally encountered in CF patients, and c) be sufficiently sensitive to detect subtle differences in fat absorption between patients with pancreatic sufficiency and those with marginal pancreatic insufficiency. This method appears to fulfill most criteria (76,77). The sensitivity of the TG*-Dy method over a wide range of steatorrhea depends on the choice of the fatty acyl moiety of the triglyceride and the positional labeling of the lipid. Recent work has determined the appropriate triglyceride to use to study a wide range of malabsorption in patients with CF and demonstrates excellent correlation with CFA (77).

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Sucrose Polybehenate

A second spot stool method has been developed employing sucrose polybehenate (SPB), which is a constituent of olestra (Olean®, Procter and Gamble, Cincinnati, OH), the nonabsorbable lipid that is currently used in a variety of foodstuffs. SPB is virtually identical with other triglycerides but is passed intact in the feces because it is not hydrolyzed by pancreatic lipase nor absorbed from the intestine. With the exception of peanut oil, behenic acid is present only in trace amounts in the normal diet (78).

Studies using SPB as a marker of fat absorption in rodents demonstrated physiologic absorption of fat from safflower oil, reductions in fat absorption with addition of olestra to safflower oil and reduced fat absorption with addition of calcium soaps (79). Initial human studies have focused on the dose and timing of appearance of a stool marker, assessing the dose of SPB needed to allow reliable identification in stool, measurement of SPB to total and individual fatty acids in stool and comparison of the SPB method to the balance method in healthy adults. Preliminary clinical studies comparing the SPB method with conventional 72-hour balance studies showed comparable absorption of fat when healthy adults were receiving a low-fat diet. Although SPB is nonabsorbable, it may affect absorption of co-ingested dietary fats; however, the small tracer amounts used in the “test dose” make this unlikely. Although this method is not yet as well validated as TG*-Dy, it shows promise. The SPB method requires only a small amount of stool and laboratory equipment commonly available in clinical and research laboratories for analysis.

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As we have described above, CF affects a variety of processes that impact on maldigestion and malabsorption (Fig. 2). Aberrant CFTR function plays a role in the entire gastrointestinal tract, not just in the pancreas and the liver. Thus, there is a great need to examine the effects of loss of intestinal CFTR beyond its role as a chloride channel. For example, insights into the pathobiology of intestinal disease in CF could be discerned by further evaluation of the direct or indirect role of CFTR on the flux or active transport of other ions such as bicarbonate and nutrients such as peptides, amino acids and glucose. The potential effects of mutant CFTR on the unstirred water layer and on intestinal mucus deserve special attention. These and other factors may all contribute to aberrant digestive and absorptive function in patients with CF. Animal models of CF show great promise to explore the intestinal consequences of loss of CFTR function or its dysfunction provided species-specific differences and the influence of diet and genetic background are accounted for.

FIG. 2

FIG. 2

Although this workshop focused primarily on lipids, maldigestion and malabsorption of other nutrients (proteins, starches, phospholipids and cholesterol) and micronutrients (vitamins, minerals and trace elements) deserve considerable attention. Nevertheless, it is likely that a better understanding of the mechanisms of deranged triglyceride digestion and absorption will lead to insight into difficulties with other nutrients.

The design and methodologies of clinical trials to determine the effectiveness of PERT deserve greater attention. Study designs should minimize risk and discomfort to subjects by validating alternative simple, sensitive methods of assessing the consequences of maldigestion and/or malabsorption, by using alternative statistical approaches to enable fewer subjects to be studied and by minimizing the use of placebos. We need to understand and dissect out the multiple interdependent and independent factors that may contribute to a true or apparent “poor response” to PERT in a substantial subset of patients with CF. More specific ways of measuring these factors will strengthen the design of studies of PERT efficacy. We require additional insight into potential confounders of the effectiveness of PERT, including, but not limited to, the discordance of gastric emptying and PERT, the site of action of PERT, the role of intestinal pH and other confounding factors such as SIBO and mucosal function. There is a compelling need to understand how bile salt composition and cycling are altered in patients with CF, what markers can be used to study this, and how CFLD contributes to malabsorption. Our understanding of the pulmonary manifestations of CF has improved dramatically as we have moved into the 21st century and these advances are being translated into better treatments and improved outcomes. Our patients should expect nothing less of our understanding and treatment of gastrointestinal manifestations of CF disease.

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Michael Wilschanski, Claude C. Roy, Steven D. Freedman, Lloyd Mayer, Birgitta Strandvik, Andrew Feranchak, Andrew E. Mulberg, Daina Kalnins, Vidar Wendel-Hansen, Susan Casey, Sarah Jane Schwarzenberg, Suntje Sander-Struckmeier, Maria R. Mascarenhas, Jonathan Cohn, Nadia Ameen, Paul Hyman.

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Jacqueline Fridge, Ruyi He, Jane A. Keng, Manon Vezina, Fathia Gibril, Friederike Henniges, Alan Kimura, Frederick T. Murray, Elizabeth A. Stafford, Marco Anelli, Yann Le Cam, Nissa Erickson, Karen Futternecht, Courtney C. Harper, Rein Sennsalu, Hugo Gallo-Torres, Tanja Gonska, Arleen Pikos, Tibor Sipos, Jason S. Soden.

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Melissa A. Ashlock, Robert J. Beall, Preston W. Campbell, Bruce Marshall, Christopher M. Penland, Diana R. Wetmore.

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