Intestinal failure (IF) exists when the intestinal mucosa is unable to absorb nutrients sufficient to sustain appropriate growth and/or physiologic balance in adults and children (1). Classically the cause of IF is lack of adequate surface area due to intestinal atresia or intestinal resection and is known as short bowel syndrome (SBS). IF also results from diminished intestinal function found in diseases such as intestinal pseudoobstruction. People with IF may require parenteral nutritional support for months to years with the potential for lifelong dependence until death or intestinal transplantation. Eventually, use of long-term parenteral nutrition (PN) leads to progressive, multifactorial liver injury frequently classified as PN-associated liver disease (PNALD). We hypothesize that some of the pathologic changes in PNALD may be secondary to inadequate intake of specific micronutrients in the diet or the PN. One such possible nutrient is choline.
Choline is a quartenary amine present in all mammalian tissues. It is an essential component of cell membranes and is required for the synthesis of phospholipids. It is also a precursor for the neurotransmitter acetylcholine and serves as a methyl group donor (2). Choline can be synthesized de novo from methionine; however, the process is most efficient when the methionine enters the liver via the portal vein. When methionine is administered parenterally, it is shunted toward metabolic pathways favoring transamidation, rendering the methyl group unavailable for the transsulfuration reactions required for choline synthesis (3–5).
Although choline was previously believed to be a nonessential nutrient because of the existence of metabolic pathways for its synthesis (6), healthy adults fed a low choline diet will develop choline deficiency as well as elevated aminotransferases and fatty infiltration of the liver, which resolve with choline supplementation (7). Additionally, adults on long-term PN supplying >80% of their nutritional needs exhibit choline deficiency, along with elevated transaminases and hepatic steatosis (8). Choline supplementation delivered intravenously in the free form (as choline chloride) or orally as a free or bound form (a salt or lipid) (9,10) is effective; the pathologic changes return when choline supplementation is stopped (11). Previous studies have also demonstrated low plasma free choline levels in children receiving PN (12) despite use of intravenous fat containing choline as a lipid moiety (8) and intravenous methionine (as a component of intravenous amino acid solutions). Choline deficiency can also worsen hepatic steatosis because adequate choline levels are required for exporting triglycerides from the liver (13). Furthermore, cirrhotic patients are unable to synthesize choline de novo despite enteral administration of elemental formulas containing methionine (14).
Children with IF are a population particularly at risk for choline deficiency secondary to commercial inavailability of an intravenous form of choline. Although enteral formulas do contain some free choline, the amount of choline delivered enterally can be less than the defined adequate intake (AI; defined in (2)). In the presence of IF there is frequently also significant malabsorption resulting from resection of small bowel and/or functional impairment. Increased dietary requirements, catheter-associated infections, and developmental changes place children with IF at greater risk for complications of choline deficiency. Choline plays a role in brain development (15,16), and plasma choline levels are significantly higher in children compared to adults (3.5-fold greater in a newborn than in an adult) (17). Studies of an endotoxin shock model in rats have shown that choline-deficient diets worsen survival after exposure to lipopolysaccharides, and a choline-supplemented diet is protective, increasing survival and decreasing aspartate aminotransferase (AST) elevations (18,19). Additionally, for adults receiving PN, choline deficiency is a risk factor for catheter thrombosis (20).
Based upon the above, we hypothesized that choline deficiency would be prevalent in a group of children with IF and amenable to correction with oral supplementation. To address this we undertook a small pilot study to ascertain whether supplementation with choline at AI dose for age was adequate for the repletion of plasma free choline levels in children.
PATIENTS AND METHODS
All children older than 40 weeks postconceptual age with IF receiving parenteral nutrition for at least 1 month and receiving enteral feeds were eligible. Patients in renal or hepatic failure were excluded. All other aspects of patient care, including aggressive advancement of enteral feeding, were unaffected by the study. This was an open label, unblinded study. Written informed consent and, where applicable, assent were obtained from all of the enrolled patients. The study was approved by the University's institutional review board.
Determination of Normal Choline Percentiles
Raw data on free choline levels versus age (from reference (17)) were kindly provided by Prof Dr Y.O. Ilcol. Due dates were used to fit percentile curves following the LMS method outlined by Rigby et al (21,22) using the R package gamlss. Diagnostics check on the model was done using worm plots (23), which indicated that the model fit the data adequately. Data allowed calculations of percentiles to 12 years old, at which point the slope of the 50th percentile had decreased to <0.3 pmol/mL/day, so values for greater than 12 years old were extended at that level.
Plasma Free Choline Measurement
A total of 1 to 2 mL of blood were drawn with regular monitoring labs into an EDTA tube and placed immediately on ice. Samples were then centrifuged at 2000g for 10 minutes at 4°C within 30 minutes of collection. The plasma was frozen at −80°C and shipped on dry ice to the University of North Carolina Nutrition Obesity Research Center. Choline, betaine, phosphatidylcholine, and sphingomyelin were extracted from plasma by the method of Bligh and Dyer (24). Aqueous and organic compounds were separated, analyzed, and quantified directly using liquid chromatography/electrospray ionization-isotope dilution mass spectrometry (LC/ESI-IDMS) after the addition of internal standards labeled with stable isotopes that are used to correct for recovery (25).
Deficient patients (defined as ≤50th percentile) were started on oral choline supplementation at AI ± 15%, defined as in reference (2), summarized in Table 1. For those less than 1 year of age, doses for both age and weight were calculated and the smaller dose was used. Choline was delivered as liquid choline chloride (Vitamin Research Products, Carson City, NV). Daily dose was divided appropriately and added to the enteral formula 2 to 4 times per day. Choline supplementation continued for 4 to 6 weeks when a second blood sample (1–2 mL) was taken for plasma choline measurement.
Alanine aminotransferase (ALT) and AST were obtained as part of routine clinical care and measured in the hospital's central laboratory.
At the first blood draw, patients received a RUQ ultrasound with a Siemens Acuson Antares Premium (Mountain View, CA) and a 6C2 probe.
Seven patients were enrolled (Table 2). Five were younger than 8 months old at the first blood draw, and 2 were 9 and 14 years old, respectively. Four patients (patients 1, 3, 4, and 7) had IF secondary to significant resection and/or high ostomy (SBS); 3 patients had functional IF (patients 2, 5, and 6). The younger patients had been on PN for 2 to 6 months, and the older patients had been on PN for several years. One patient (patient 7) died of an apparent septic event at an outside hospital near the end of the supplementation phase. Patient characteristics are summarized in Table 2.
AST and ALT are listed in Table 2. In the 6 patients for whom pre- and postsupplementation values were obtained, ALT and/or AST were abnormal at the start of supplementation in all of them. Transaminases improved in 3 of the 6 (patients 4, 5, and 6), remained essentially stable in 2 (patients 1 and 3), and worsened in 1 (patient 2).
All of the ultrasounds showed normal echogenicity.
Free Choline Percentiles Versus Age
We calculated percentiles for free choline versus age using raw data in the reference to provide meaningful comparison for the plasma free choline levels in children with IF (17). Percentile curves are plotted in Fig. 1. Free choline is sharply higher in newborns, with the 50th percentile being approximately 3.5 times that of adolescents. The level drops quickly to approximately 2-fold that of adolescents at 2 months and 1.5 times at 8.5 months. A table of percentiles for free choline versus age is available in the online-only Supplementary Table 1 (http://links.lww.com/MPG/A43).
Free Choline Levels
Before supplementation, free plasma choline percentiles were less than the third percentile for all of the patients except 1 infant (patient 5, just above the third percentile) and the 13-year-old child (patient 1, 25th to 50th percentile). The older children responded to the choline supplementation with meaningful increases in their choline percentiles. The infants, however, failed to have meaningful increases in their plasma free choline, with levels actually decreasing during supplementation in 2 infants (patients 5 and 6). Choline levels before and after supplementation are overlaid on a plot of free choline percentiles versus age (Fig. 1).
For the first time we have generated percentiles for plasma free choline stratified by age. In this population of infants with IF, supplementation with enteral choline at AI doses failed to produce meaningful increases in plasma free choline levels. In two infants, plasma levels decreased after supplementation. One of these infants had cystic fibrosis (CF) in addition to IF. Increased fecal excretion of choline-containing phospholipids has been described in children with CF (26); however, our study supplemented with free choline, so fat malabsorption should not have played a role. Increased membrane turnover of choline-containing lipids has also been shown in the platelets and fibroblasts of patients with CF (27), which may partially explain the decrease. Another study has shown improvement in abnormal plasma methionine-homocysteine metabolites and glutathione status in children with CF when choline is supplemented; however, this study used significantly higher doses of choline than AI, supplementing with twice the tolerable upper intake level for 1- to 8-year-olds (2,28). The 2 infants whose free choline did not decrease during the course of the study had the greatest decrease in percent of total energy delivered parenterally, and the only infant (patient 4) whose free choline increased to more than the third percentile with supplementation went from nearly totally dependent on PN to completely enterally fed.
Among the older children with IF, supplementation was more successful, with increases in plasma free choline levels. It is not clear from this study whether this is a result of age or intestinal function. Alternatively, it may simply be a function of choline dose, because they were receiving significantly higher doses. Taken together, the infant and adolescent data suggest the low plasma choline level reflects overall malabsorption secondary to decreased bowel surface area, although we cannot rule out deficiency due to increased turnover or excretion.
For the 6 patients in whom pre- and postsupplementation values were obtained, AST and ALT improved in half the patients. The 2 patients with essentially stable transaminases during supplementation were the adolescent patients, 1 of whom had only marginally elevated AST with a normal ALT. In 2 patients, AST and ALT normalized during the course of the study (patients 4 and 5); however, the plasma choline level increased in one and decreased in another. Given that ALT and AST can be affected by multiple processes and all of the patients were supplemented with choline with aggressive advancement of enteral feedings, the influence of choline supplementation on the improvements seen in these 3 patients is unclear and will require further placebo-controlled studies.
Interestingly, all of the children had normal hepatic echogenicity by ultrasound despite low choline levels. Recent studies have shown that ultrasound has an 80% to 90% sensitivity for detecting hepatic steatosis, but that 33% fat is the optimal threshold for detecting steatosis (29,30). Our subjects may have had low levels of steatosis, or children may be less susceptible to the development of steatosis than choline-deficient adults, or it requires a longer duration of choline deficiency than we observed in this study. Although not performed in this study, more subtle improvements in liver function with choline supplementation may be appreciated with 13C-methionine breath testing (31). Further studies with enteral supplementation of choline in infants should consider doses significantly above published AI.
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