A Controlled, Randomized, Double-Blind Trial of Prophylaxis Against Jaundice Among Breastfed Newborns
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《小儿科》
Department of Pediatrics, Oregon Health and Science University, Portland, Oregon
Department of Biostatistics and Medical Informatics
Waisman Center, University of Wisconsin, Madison, Wisconsin
ABSTRACT
Objectives. Neonatal jaundice is a greater problem for infants fed breast milk, compared with formula. This study tested the hypotheses that feeding breastfed newborns -glucuronidase inhibitors during the first week after birth would increase fecal bilirubin excretion and would reduce jaundice without affecting breastfeeding deleteriously.
Methods. Sixty-four breastfed newborns were randomized to 4 groups, ie, control or receiving 6 doses per day (5 mL per dose) of L-aspartic acid, enzymatically hydrolyzed casein (EHC), or whey/casein (W/C) for the first week. L-Aspartic acid and EHC inhibit -glucuronidase. Transcutaneous bilirubin levels (primary outcome) were measured daily (Jaundice Meter [Minolta/Air Shields, Hatboro, PA] and Bilicheck [Respironics, Pittsburgh, PA]). All stools were collected, and fecal bile pigments, including bilirubin diglucuronide, bilirubin monoglucuronides, and bilirubin, were analyzed with high-performance liquid chromatography. Follow-up assessments included day 7 body weight, day 6/7 prebreastfeeding/postbreastfeeding weights, maternal ratings, and ages at formula introduction and breastfeeding cessation.
Results. The groups were comparable at entry. Overall, the L-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels than did the control group (75.8%, 69.6%, and 69.2%, respectively, of the control mean, 8.53 mg/dL, at the bilirubin peak on day 4). The L-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels on days 3 to 7. Fecal bile pigment excretion was greatest in the L-aspartic acid group, significantly greater than control values. There were no significant differences in dosages, follow-up measurements, and maternal ratings.
Conclusions. Use of minimal aliquots of L-aspartic acid and EHC for -glucuronidase inhibition results in increased fecal bilirubin excretion and less jaundice, without disruption of the breastfeeding experience. Decreased jaundice in the W/C group, which lacked a -glucuronidase inhibitor, suggests a different mechanism.
Key Words: neonate jaundice hyperbilirubinemia breastfeeding -glucuronidase bilirubin
Abbreviations: EHC, enzymatically hydrolyzed casein W/C, whey/casein HPLC, high-performance liquid chromatography BMG, bilirubin monoglucuronide BDG, bilirubin diglucuronide BP, bile pigment AAP, American Academy of Pediatrics
Most newborns experience benign hyperbilirubinemia. Severe elevation of serum bilirubin levels, however, can result in brain damage known as kernicterus.1,2 Infants with hyperbilirubinemia may require bilirubin surveillance, feeding changes, phototherapy, or exchange transfusion, and the National Institutes of Health support development of an investigational drug that inhibits the production of bilirubin.3,4
Breastfeeding is the best choice for infant nutrition,5,6 and the incidence of breastfeeding initiation in the United States is 70%.7 Breastfed infants, however, have higher serum bilirubin concentrations than formula-fed infants,8 and 98% of the infants in a US kernicterus registry were breastfed.9 One third of those in the kernicterus registry lack a diagnosis other than breastfeeding to explain severe hyperbilirubinemia. In a large retrospective study, breastfeeding was the second highest independent factor (odds ratio: 5.7) associated with serum bilirubin concentrations of 25 mg/dL (428 μmol/L).10 In 2001, the Joint Commission on Accreditation of Healthcare Organizations,11 the Centers for Disease Control and Prevention,12 and the American Academy of Pediatrics (AAP)13 all noted increased reports of kernicterus and warned that breastfeeding is an important risk factor for the development of severe hyperbilirubinemia.
-Glucuronidase is a factor in neonatal jaundice, because it potentiates the enterohepatic circulation of bilirubin by deconjugating intestinal bilirubin conjugates, producing bilirubin that is better absorbed by the intestine.14 In utero, this facilitates bilirubin clearance via the placenta. After birth, however, the enterohepatic circulation of bilirubin delays bilirubin clearance.15 Breast milk is rich in -glucuronidase.14 Routine infant formula has negligible -glucuronidase, and infants consuming such formula have less jaundice than breastfed infants.16,17 Casein hydrolysate formula inhibits -glucuronidase, and infants consuming such formula have less jaundice than infants receiving routine formula.16,17 The major -glucuronidase inhibitor in casein hydrolysate is L-aspartic acid.18 Inhibition of human milk -glucuronidase has been shown to decrease intestinal absorption of bilirubin in a rat model with bile duct and duodenal catheters.19 The purpose of this study was to test the hypotheses that feeding breastfed newborns -glucuronidase inhibitors during the first 1 week after birth would increase fecal bilirubin excretion and would result in less jaundice without deleteriously affecting breastfeeding.
METHODS
Study Design
This project was approved by human subjects committees at the University of Wisconsin (Madison, WI) and Monroe Hospital (Monroe, WI). Enrollment extended from April 2000 to April 2002, with follow-up monitoring to August 2002. Pregnant women at Monroe Clinic, recruited through posters, contacted Dr Gourley, who obtained written consent. Consented newborns who met the following criteria were enrolled soon after birth: (1) healthy full-term (>37 weeks of gestation), (2) white (to avoid racial variables), and (3) exclusive breastfeeding planned. A formal sample size calculation was performed, in which a difference of 2 mg/dL was determined to be the effect size that we wished to detect and that might be clinically important. The estimated SD for group mean transcutaneous bilirubin levels, based on our preliminary data, was 0.87. With an of 0.05/6 = .0083 (Bonferroni adjustment) and a power of 0.85 to 0.95, the estimated group number was 15. Sixty-nine newborns were assigned (randomization table) to 1 of 4 groups, ie, breastfeeding only (control, 19 subjects) or breastfeeding plus 6 doses per day (5 mL per dose) of 1 of 3 nutritional ingredients, ie, L-aspartic acid (180 mg/5 mL of water) (17 subjects), enzymatically hydrolyzed casein (EHC) (11.9 g plus 88.1 g of water; 10% protein equivalent solution with 0.5 g of nitrogen per 5 mL) (16 subjects), or whey/casein (W/C) (60/40; 6 g of whey protein isolate plus 4 g of sodium caseinate from cow milk plus 89 g of water; 10% protein with 0.5 g of protein per 5 mL) (17 subjects). The W/C group was included as another type of control group (ie, receiving no -glucuronidase inhibitor) but, unlike the control group, which received nothing but breastfeeding, the W/C group received a small amount of protein that lacked any -glucuronidase inhibitor. These dosages were determined on the basis of prior studies with formula-fed infants.16,17 Six newborns withdrew (1 in the control group, because of infant illness; 2 in the L-aspartic acid group, because 1 received formula early and 1 received formula on the fourth day [therefore, only data before this were included in the analysis]; 1 in the EHC group, because the mother rejected breastfeeding; and 2 in the W/C group, because of maternal illness and maternal dislike of W/C odor). Doses, beginning with the second breastfeeding, were administered when the infant was changing breasts or after breastfeeding, with an oral syringe that was preloaded daily by a study nurse who visited the patient in the hospital and at home, collected data, provided similar breastfeeding education/support to all groups, and reported adverse reactions.
Parents and researchers were blinded to the treatment identity. The code was not broken until study completion. Mead Johnson (Evansville, IN) prepared the code and ingredients (components of current infant formulas) and confirmed product sterility throughout the study.
Outcome Measures
Data confirming group comparability included gender, gestational age, birth weight, and delivery with forceps, vacuum extraction, or cesarean section. Daily transcutaneous jaundice index values and bilirubin levels were measured (forehead) with a Jaundice Meter-102 (Minolta/Air Shields, Hatboro, PA; 3 readings were averaged)20 and a Bilicheck (Respironics, Pittsburgh, PA; 1 reading included 5 replicates),21,22 respectively (primary outcome measures). Hospital and home jaundice measurements were usually made between the hours of 9 AM and 5 PM.
All stools were collected by saving diapers in a freezer and recording the date and time of stool passage, pooled for each 24-hour period after birth, and analyzed for bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG), bilirubin, total bile pigments (BPs) (BDG plus BMG plus bilirubin), urobilinoids, and zinc coproporphyrin (a meconium marker) with reverse-phase high-performance liquid chromatography (HPLC), as described previously,23 with the following modifications. After BPs were extracted into the upper organic layer, this layer was removed and pooled with a triple extraction of the protein interface. This extraction was accomplished by removing the interface, adding 0.1 mL of dimethylformamide, sonicating the mixture for 1 minute, and centrifuging the mixture (12400g for 1 minute). This pooled sample (0.5 mL) was refrigerated (–20°C for 15 minutes) to precipitate protein and recentrifuged (12700g for 5 minutes), the clarified organic extract was transferred to a 0.22-μm nylon microfuge filter (MSI, Westboro, MA) and recentrifuged (6370g for 2 minutes), and 20 μL were analyzed with a Hewlett Packard model 1090 HPLC system (Hewlett Packard, Brookfield, WI) with a 20-mm, C18 μBondapak guard column (Waters Associates, Milford, MA). Detection was at 436 nm (bandwidth: 4 nm) minus 554 nm (bandwidth: 22 nm). Because the extinction coefficients for bilirubin, BMG, and BDG are nearly identical,24 the standard curve for bilirubin was used to quantify these pigments with a coumarin 6 internal standard.
Urobilinoids were determined by oxidizing urobilinogens to urobilins and were measured as 1 group.25 Potassium iodate ensured complete oxidation; stercobilin and fluorescein served as standards, with results being expressed in stercobilin equivalents.26
Daily fecal output included wet and dry weights, allowing quantitation of total excretion. Parents logged feeding and dosage times. Doses received were recorded (all, most, or little). On days 6 and 7, parents weighed their infants before and after breastfeeding (BLB-12 scale; Tanita Corp, Tokyo, Japan), to determine intake, as frequently as they considered comfortable. Study termination assessment included a nude weight.
All infants were exclusively breastfed except for study doses. Parents completed a long-term follow-up questionnaire after the study, including age of first nonstudy supplementation, age of breastfeeding cessation, and impression of study (excellent, good, satisfactory, poor, and very poor). Several subjects were unreachable for long-term follow-up assessment (2 of 18 in the control group, 4 of 16 in the L-aspartic acid group, 1 of 15 in the EHC group, and 2 of 15 in the W/C group).
Statistical Methods
Group characteristics and follow-up information were compared with analyses of variance and 2 tests for continuous and discrete variables, respectively. Jaundice was assessed daily with the Bilicheck and Jaundice Meter, and the time of measurement was recorded. Because the units of measurement differed, a regression equation was developed to estimate transcutaneous bilirubin levels from jaundice indices on the basis of all data (Bilicheck data, Jaundice Meter data, and time). The average transcutaneous bilirubin level from both meters for each subject on each day was used to represent the transcutaneous bilirubin level.
Repeated measures of transcutaneous bilirubin levels, fecal BPs [normalized as log(measurements + 1)], and dosages among groups were compared with a generalized estimating equation27 with an independence working correlation and an identity link adjusting for time to a third-order polynomial. Models were evaluated by examining predicted versus residual values, and 2 extreme outliers were dropped from 501 bilirubin measurements. When there were significant overall model effects (P < .05), paired group comparisons were made and results were considered significant at P < .0083 (0.05/6; Bonferroni adjustment).28,29 Paired comparisons were made each day, and results were considered significant at P < .0083 (Bonferroni adjustment).28,29 Our methods were detailed previously.17
RESULTS
There were no significant differences in gender, birth weight, vacuum extraction rate, and cesarean section rate and no forceps deliveries (Table 1). Although they were statistically significant, the gestational age differences were not clinically significant.
We also performed analyses of fecal excretion of BDG, BMG, and bilirubin. Overall BDG fecal excretion was significantly greater than control values in the EHC (P = .0025) and W/C (P = .0053) groups. Daily comparisons showed that the EHC group excreted more fecal BDG than did the control group on days 3 to 5 and the W/C group excreted more BDG than did the control group on days 2 to 4. Paired daily comparisons showed that the 3 treatment groups did not differ from each other in BDG excretion. Overall fecal BMG excretion was significantly greater than control values in all treatment groups (P < .001 for all), and the L-aspartic acid and EHC groups did not differ significantly in BMG excretion from the W/C group. The L-aspartic acid and EHC groups excreted more fecal BMG than did the control group on days 2 to 6, and the W/C group excreted more BMG than did the control group on days 2 to 4. Paired daily comparisons showed that the L-aspartic acid group excreted more BMG than did the W/C group on days 5 to 6. Overall fecal bilirubin excretion was significantly higher than control values in the L-aspartic acid (P < .001) and W/C (P = .0022) groups. The L-aspartic acid group excreted more fecal bilirubin than did the control group on days 4 to 6, and the W/C group excreted more bilirubin than did the control group on days 2 to 4. Fecal BP excretion resembled bilirubin excretion because the magnitudes of BDG and BMG fecal excretion were only 0.13% and 21% that of bilirubin, respectively. BDG, BMG, and bilirubin fecal excretion did not differ significantly between the L-aspartic acid and EHC groups. Dosages received among the treatment groups showed no significant differences in number of doses per day (P = .42), number of feedings per day (P = .74), total dosage volume per day (P = .80), or percentage of theoretical cumulative dose received (P = .99).
Follow-up information showed no significant differences in day 7 weight, number of day 6/7 weighed feedings, weight of day 6/7 milk and treatment, weighed day 6/7 milk, age at first formula feeding, breastfeeding cessation age, and mother’s rating of at least good (Table 2). There were no significant differences among the groups in meconium clearance, as determined with fecal zinc coproporphyrin measurements (P = .59), or in fecal urobilinoid levels (P = .43). There were no known adverse events or side effects. None of the infants demonstrated excessive weight loss (ie, >10%).
DISCUSSION
Our results showed that breastfed newborns given L-aspartic acid, EHC, or W/C during the first week had significantly lower transcutaneous bilirubin levels than did control subjects. We hypothesized that feeding -glucuronidase inhibitors (L-aspartic acid or EHC) would inhibit -glucuronidase present in the intestine. -Glucuronidase (-G) hydrolyzes glucuronic acid from bilirubin conjugates, converting BDG to BMG and BMG to bilirubin as illustrated.
If -glucuronidase is inhibited, then there should be relative preservation of conjugates on the left side of this equation. The fate of bilirubin is more complicated, however, because bilirubin is more easily absorbed from the intestine than are bilirubin conjugates30,31 and because intestinal bacteria can convert bilirubin and its conjugates to urobilinoids, a family of reduction-oxidation products.32 If bilirubin is not absorbed or converted to urobilinoids, then it is excreted in feces. The EHC group excreted significantly more fecal BDG and BMG than did the control group. The L-aspartic acid group also excreted significantly more BMG than did the control group. Interestingly, the W/C group also excreted more BDG and BMG than did the control group. Because W/C lacks -glucuronidase inhibitors, some other mechanism must underlie this observation. One possibility is increased binding of bilirubin conjugates by components of the W/C supplement in the intestinal lumen. Overall, however, fecal excretion of bilirubin glucuronides was minor, compared with total fecal bilirubin excretion (predominantly unconjugated). This underscores the importance of -glucuronidase, because little unconjugated bilirubin is excreted into bile. Bilirubin excretion was significantly higher in the L-aspartic acid group, compared with the control group, consistent with inhibition of -glucuronidase in the L-aspartic acid group.
Important events occur at the time of birth regarding bilirubin metabolism. Bilirubin glucuronides are present in fetal bile by 22 weeks of gestation and increase in concentration with the maturation of hepatic bilirubin glucuronosyltransferase.33 In the intestinal lumen, bilirubin glucuronides can undergo hydrolysis by -glucuronidase, producing bilirubin that is more easily reabsorbed into the circulation.30,31 In the fetus, this mechanism facilitates placental clearance of bilirubin. After birth, however, this enterohepatic bilirubin circulation delays clearance and prolongs jaundice. Although bilirubin glucuronosyltransferase does not plateau at adult levels until 14 weeks of age,34 the small but statistically significant gestational age differences among our groups cannot explain the findings in this study, because only the L-aspartic acid group had a mean gestational age greater than that of the control group.
An important consideration is any effect on breastfeeding. The AAP promotes breastfeeding as the best infant nutrition5,6 and recommends against supplements (eg, water, glucose-water solution, or formula) unless a medical indication exists.6 The AAP Clinical Practice Guideline regarding neonatal hyperbilirubinemia recommends against routine supplementation of nondehydrated breastfed infants with water or dextrose-water solution, noting that this does not prevent hyperbilirubinemia. It also lists exclusive breastfeeding as a major risk factor for the development of severe hyperbilirubinemia and exclusive bottle-feeding as a factor associated with decreased risk of significant jaundice.2 In the management of jaundiced breastfed infants, the AAP notes that temporary interruption of breastfeeding, with the substitution of formula, can reduce bilirubin levels; if supplemental fluid is needed, then the best fluid to use is a milk-based formula, because it inhibits the enterohepatic circulation of bilirubin and should help lower serum bilirubin levels. Our data showed that feeding of L-aspartic acid, EHC, or W/C in volumes of up to 30 mL per day had no deleterious effect on weight gain, milk intake per feeding, age at subsequent first supplementation, or duration of breastfeeding. Mothers approved the feedings, with 100% rating their study experience as good or excellent in the control, L-aspartic acid, and EHC groups. Estimated energy intake attributable to study nutrients was negligible (<50 J/day, <13.8 J/kg of birth weight per day). Our experience was similar to that of Nicoll et al,35 who showed that 38.6 mL of 5% dextrose per 1 kg of birth weight per day had no deleterious effect on breastfeeding, and our study used much less.
As judged by the age at first supplementation and the age at breastfeeding cessation, this study included many highly motivated, breastfeeding mothers. However, mothers could benefit from this approach independent of breastfeeding duration, because the approach was limited to the first week of life. The interventions were simple and well tolerated, as verified by the high ratings parents gave the study after completion. As indicated in Table 2, mothers rated this study very highly, and it is unlikely they would do so if these interventions were problematic. Therefore, we think that such interventions can be quite acceptable to both mothers and pediatricians, as part of a breastfeeding support/education program.
The 2004 AAP hyperbilirubinemia guidelines2 include a nomogram developed by Bhutani et al36 to classify serum bilirubin concentrations into 4 different risk zones. Seventeen of our subjects were in a risk zone above the low-risk zone within the first 48 hours after birth (6 in the control group, 5 in the L-aspartic acid group, 1 in the EHC group, and 5 in the W/C group). The decline times (in hours) from a risk zone above the low-risk zone within the first 48 hours to a low-risk zone after 48 hours were compared among the 4 groups for these 17 subjects. There were no significant differences in decline times between the 2 groups that received no -glucuronidase inhibitor (control: mean ± SD: 49.00 ± 34.01 hours; median: 31.00 hours; W/C: mean ± SD: 48.90 ± 5.70 hours; median: 49.58 hours; P = .41). There were also no significant differences in decline times between the 2 groups that received a -glucuronidase inhibitor (L-aspartic acid: mean ± SD: 25.23 ± 16.84 hours; median: 14.67 hours; EHC: mean ± SD: 23.58 ± 0 hours; median: 23.58 hours; P = 1.0). The combined inhibitor groups had a significantly shorter decline time than the combined no-inhibitor groups (inhibitor: mean ± SD: 24.96 ± 15.08 hours; median: 19.13 hours; no-inhibitor: mean ± SD: 48.95 ± 24.32 hours; median: 45.33 hours; P = .039, Wilcoxon test) (Fig 3). Only 1 subject in our entire study was in the highest (>95th percentile) risk zone (age: 22.7 hours). That subject was in the EHC group, and the risk decreased to the next-to-lowest risk zone (40–75th percentile) at 46.3 hours and to the lowest risk zone at 74.9 hours. It would be of interest to evaluate the effect of -glucuronidase inhibitors on larger numbers of infants in high-risk zones.
Although 64 infants were recruited into the study, when the infants were randomized into 4 groups each group became rather small. Nevertheless, our study showed significant differences clearly. Because this was a controlled, randomized, double-blind trial, we have confidence in its real clinical significance. However, future studies with larger sample sizes are warranted. Our treatment groups had peak bilirubin levels 25% to 30% lower than control values, and those that received the -glucuronidase inhibitor did decrease to lower risk zones faster than the groups that did not receive the -glucuronidase inhibitor. Whether this would be helpful for infants with higher bilirubin levels is not addressed with these data. However, if infants with bilirubin levels in the high-risk zone decreased their levels by 25% to 30%, this certainly could be helpful.
It was the hope of this study that breastfed infants could have the best of both worlds, ie, the advantages of exclusive breastfeeding plus the decreased risk of severe hyperbilirubinemia associated with exclusive formula feeding. The results show that significant reductions in transcutaneous bilirubin levels can be achieved, with no negative effect on breastfeeding, through administration of small daily amounts of selected key ingredients of currently available infant formula. In the L-aspartic acid and EHC groups, the effect probably results from -glucuronidase inhibition and increased fecal excretion of bilirubin. Lower transcutaneous bilirubin levels in the W/C group suggest a different mechanism. These results require confirmation with additional research involving larger numbers of subjects.
ACKNOWLEDGMENTS
This work was supported in part by grants from Mead Johnson Nutritionals, the University of Wisconsin Technology and Innovation Fund, and the University of Wisconsin Department of Pediatrics and Medical School.
Sincere thanks go to the following nurses: Mona Borgman, Sarah Goebel, Gynel Hagemann, Carmen McDonald, and Deb Wolff. Thanks also go to Deb Kundert and Pat Berra. We are greatly indebted to Dr Bruce Duemler, his co-workers, and the staff of the Monroe Hospital and Clinic (Monroe, WI), who embraced and supported this study enthusiastically, and to Dr John D. Bancroft. Thanks go to Scott Kerr, Respironics, for providing Bilicheck devices. This manuscript is dedicated to the memory of Dr Gerard B. Odell.
FOOTNOTES
Accepted Nov 23, 2004.
No conflict of interest declared.
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Department of Biostatistics and Medical Informatics
Waisman Center, University of Wisconsin, Madison, Wisconsin
ABSTRACT
Objectives. Neonatal jaundice is a greater problem for infants fed breast milk, compared with formula. This study tested the hypotheses that feeding breastfed newborns -glucuronidase inhibitors during the first week after birth would increase fecal bilirubin excretion and would reduce jaundice without affecting breastfeeding deleteriously.
Methods. Sixty-four breastfed newborns were randomized to 4 groups, ie, control or receiving 6 doses per day (5 mL per dose) of L-aspartic acid, enzymatically hydrolyzed casein (EHC), or whey/casein (W/C) for the first week. L-Aspartic acid and EHC inhibit -glucuronidase. Transcutaneous bilirubin levels (primary outcome) were measured daily (Jaundice Meter [Minolta/Air Shields, Hatboro, PA] and Bilicheck [Respironics, Pittsburgh, PA]). All stools were collected, and fecal bile pigments, including bilirubin diglucuronide, bilirubin monoglucuronides, and bilirubin, were analyzed with high-performance liquid chromatography. Follow-up assessments included day 7 body weight, day 6/7 prebreastfeeding/postbreastfeeding weights, maternal ratings, and ages at formula introduction and breastfeeding cessation.
Results. The groups were comparable at entry. Overall, the L-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels than did the control group (75.8%, 69.6%, and 69.2%, respectively, of the control mean, 8.53 mg/dL, at the bilirubin peak on day 4). The L-aspartic acid, EHC, and W/C groups had significantly lower transcutaneous bilirubin levels on days 3 to 7. Fecal bile pigment excretion was greatest in the L-aspartic acid group, significantly greater than control values. There were no significant differences in dosages, follow-up measurements, and maternal ratings.
Conclusions. Use of minimal aliquots of L-aspartic acid and EHC for -glucuronidase inhibition results in increased fecal bilirubin excretion and less jaundice, without disruption of the breastfeeding experience. Decreased jaundice in the W/C group, which lacked a -glucuronidase inhibitor, suggests a different mechanism.
Key Words: neonate jaundice hyperbilirubinemia breastfeeding -glucuronidase bilirubin
Abbreviations: EHC, enzymatically hydrolyzed casein W/C, whey/casein HPLC, high-performance liquid chromatography BMG, bilirubin monoglucuronide BDG, bilirubin diglucuronide BP, bile pigment AAP, American Academy of Pediatrics
Most newborns experience benign hyperbilirubinemia. Severe elevation of serum bilirubin levels, however, can result in brain damage known as kernicterus.1,2 Infants with hyperbilirubinemia may require bilirubin surveillance, feeding changes, phototherapy, or exchange transfusion, and the National Institutes of Health support development of an investigational drug that inhibits the production of bilirubin.3,4
Breastfeeding is the best choice for infant nutrition,5,6 and the incidence of breastfeeding initiation in the United States is 70%.7 Breastfed infants, however, have higher serum bilirubin concentrations than formula-fed infants,8 and 98% of the infants in a US kernicterus registry were breastfed.9 One third of those in the kernicterus registry lack a diagnosis other than breastfeeding to explain severe hyperbilirubinemia. In a large retrospective study, breastfeeding was the second highest independent factor (odds ratio: 5.7) associated with serum bilirubin concentrations of 25 mg/dL (428 μmol/L).10 In 2001, the Joint Commission on Accreditation of Healthcare Organizations,11 the Centers for Disease Control and Prevention,12 and the American Academy of Pediatrics (AAP)13 all noted increased reports of kernicterus and warned that breastfeeding is an important risk factor for the development of severe hyperbilirubinemia.
-Glucuronidase is a factor in neonatal jaundice, because it potentiates the enterohepatic circulation of bilirubin by deconjugating intestinal bilirubin conjugates, producing bilirubin that is better absorbed by the intestine.14 In utero, this facilitates bilirubin clearance via the placenta. After birth, however, the enterohepatic circulation of bilirubin delays bilirubin clearance.15 Breast milk is rich in -glucuronidase.14 Routine infant formula has negligible -glucuronidase, and infants consuming such formula have less jaundice than breastfed infants.16,17 Casein hydrolysate formula inhibits -glucuronidase, and infants consuming such formula have less jaundice than infants receiving routine formula.16,17 The major -glucuronidase inhibitor in casein hydrolysate is L-aspartic acid.18 Inhibition of human milk -glucuronidase has been shown to decrease intestinal absorption of bilirubin in a rat model with bile duct and duodenal catheters.19 The purpose of this study was to test the hypotheses that feeding breastfed newborns -glucuronidase inhibitors during the first 1 week after birth would increase fecal bilirubin excretion and would result in less jaundice without deleteriously affecting breastfeeding.
METHODS
Study Design
This project was approved by human subjects committees at the University of Wisconsin (Madison, WI) and Monroe Hospital (Monroe, WI). Enrollment extended from April 2000 to April 2002, with follow-up monitoring to August 2002. Pregnant women at Monroe Clinic, recruited through posters, contacted Dr Gourley, who obtained written consent. Consented newborns who met the following criteria were enrolled soon after birth: (1) healthy full-term (>37 weeks of gestation), (2) white (to avoid racial variables), and (3) exclusive breastfeeding planned. A formal sample size calculation was performed, in which a difference of 2 mg/dL was determined to be the effect size that we wished to detect and that might be clinically important. The estimated SD for group mean transcutaneous bilirubin levels, based on our preliminary data, was 0.87. With an of 0.05/6 = .0083 (Bonferroni adjustment) and a power of 0.85 to 0.95, the estimated group number was 15. Sixty-nine newborns were assigned (randomization table) to 1 of 4 groups, ie, breastfeeding only (control, 19 subjects) or breastfeeding plus 6 doses per day (5 mL per dose) of 1 of 3 nutritional ingredients, ie, L-aspartic acid (180 mg/5 mL of water) (17 subjects), enzymatically hydrolyzed casein (EHC) (11.9 g plus 88.1 g of water; 10% protein equivalent solution with 0.5 g of nitrogen per 5 mL) (16 subjects), or whey/casein (W/C) (60/40; 6 g of whey protein isolate plus 4 g of sodium caseinate from cow milk plus 89 g of water; 10% protein with 0.5 g of protein per 5 mL) (17 subjects). The W/C group was included as another type of control group (ie, receiving no -glucuronidase inhibitor) but, unlike the control group, which received nothing but breastfeeding, the W/C group received a small amount of protein that lacked any -glucuronidase inhibitor. These dosages were determined on the basis of prior studies with formula-fed infants.16,17 Six newborns withdrew (1 in the control group, because of infant illness; 2 in the L-aspartic acid group, because 1 received formula early and 1 received formula on the fourth day [therefore, only data before this were included in the analysis]; 1 in the EHC group, because the mother rejected breastfeeding; and 2 in the W/C group, because of maternal illness and maternal dislike of W/C odor). Doses, beginning with the second breastfeeding, were administered when the infant was changing breasts or after breastfeeding, with an oral syringe that was preloaded daily by a study nurse who visited the patient in the hospital and at home, collected data, provided similar breastfeeding education/support to all groups, and reported adverse reactions.
Parents and researchers were blinded to the treatment identity. The code was not broken until study completion. Mead Johnson (Evansville, IN) prepared the code and ingredients (components of current infant formulas) and confirmed product sterility throughout the study.
Outcome Measures
Data confirming group comparability included gender, gestational age, birth weight, and delivery with forceps, vacuum extraction, or cesarean section. Daily transcutaneous jaundice index values and bilirubin levels were measured (forehead) with a Jaundice Meter-102 (Minolta/Air Shields, Hatboro, PA; 3 readings were averaged)20 and a Bilicheck (Respironics, Pittsburgh, PA; 1 reading included 5 replicates),21,22 respectively (primary outcome measures). Hospital and home jaundice measurements were usually made between the hours of 9 AM and 5 PM.
All stools were collected by saving diapers in a freezer and recording the date and time of stool passage, pooled for each 24-hour period after birth, and analyzed for bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG), bilirubin, total bile pigments (BPs) (BDG plus BMG plus bilirubin), urobilinoids, and zinc coproporphyrin (a meconium marker) with reverse-phase high-performance liquid chromatography (HPLC), as described previously,23 with the following modifications. After BPs were extracted into the upper organic layer, this layer was removed and pooled with a triple extraction of the protein interface. This extraction was accomplished by removing the interface, adding 0.1 mL of dimethylformamide, sonicating the mixture for 1 minute, and centrifuging the mixture (12400g for 1 minute). This pooled sample (0.5 mL) was refrigerated (–20°C for 15 minutes) to precipitate protein and recentrifuged (12700g for 5 minutes), the clarified organic extract was transferred to a 0.22-μm nylon microfuge filter (MSI, Westboro, MA) and recentrifuged (6370g for 2 minutes), and 20 μL were analyzed with a Hewlett Packard model 1090 HPLC system (Hewlett Packard, Brookfield, WI) with a 20-mm, C18 μBondapak guard column (Waters Associates, Milford, MA). Detection was at 436 nm (bandwidth: 4 nm) minus 554 nm (bandwidth: 22 nm). Because the extinction coefficients for bilirubin, BMG, and BDG are nearly identical,24 the standard curve for bilirubin was used to quantify these pigments with a coumarin 6 internal standard.
Urobilinoids were determined by oxidizing urobilinogens to urobilins and were measured as 1 group.25 Potassium iodate ensured complete oxidation; stercobilin and fluorescein served as standards, with results being expressed in stercobilin equivalents.26
Daily fecal output included wet and dry weights, allowing quantitation of total excretion. Parents logged feeding and dosage times. Doses received were recorded (all, most, or little). On days 6 and 7, parents weighed their infants before and after breastfeeding (BLB-12 scale; Tanita Corp, Tokyo, Japan), to determine intake, as frequently as they considered comfortable. Study termination assessment included a nude weight.
All infants were exclusively breastfed except for study doses. Parents completed a long-term follow-up questionnaire after the study, including age of first nonstudy supplementation, age of breastfeeding cessation, and impression of study (excellent, good, satisfactory, poor, and very poor). Several subjects were unreachable for long-term follow-up assessment (2 of 18 in the control group, 4 of 16 in the L-aspartic acid group, 1 of 15 in the EHC group, and 2 of 15 in the W/C group).
Statistical Methods
Group characteristics and follow-up information were compared with analyses of variance and 2 tests for continuous and discrete variables, respectively. Jaundice was assessed daily with the Bilicheck and Jaundice Meter, and the time of measurement was recorded. Because the units of measurement differed, a regression equation was developed to estimate transcutaneous bilirubin levels from jaundice indices on the basis of all data (Bilicheck data, Jaundice Meter data, and time). The average transcutaneous bilirubin level from both meters for each subject on each day was used to represent the transcutaneous bilirubin level.
Repeated measures of transcutaneous bilirubin levels, fecal BPs [normalized as log(measurements + 1)], and dosages among groups were compared with a generalized estimating equation27 with an independence working correlation and an identity link adjusting for time to a third-order polynomial. Models were evaluated by examining predicted versus residual values, and 2 extreme outliers were dropped from 501 bilirubin measurements. When there were significant overall model effects (P < .05), paired group comparisons were made and results were considered significant at P < .0083 (0.05/6; Bonferroni adjustment).28,29 Paired comparisons were made each day, and results were considered significant at P < .0083 (Bonferroni adjustment).28,29 Our methods were detailed previously.17
RESULTS
There were no significant differences in gender, birth weight, vacuum extraction rate, and cesarean section rate and no forceps deliveries (Table 1). Although they were statistically significant, the gestational age differences were not clinically significant.
We also performed analyses of fecal excretion of BDG, BMG, and bilirubin. Overall BDG fecal excretion was significantly greater than control values in the EHC (P = .0025) and W/C (P = .0053) groups. Daily comparisons showed that the EHC group excreted more fecal BDG than did the control group on days 3 to 5 and the W/C group excreted more BDG than did the control group on days 2 to 4. Paired daily comparisons showed that the 3 treatment groups did not differ from each other in BDG excretion. Overall fecal BMG excretion was significantly greater than control values in all treatment groups (P < .001 for all), and the L-aspartic acid and EHC groups did not differ significantly in BMG excretion from the W/C group. The L-aspartic acid and EHC groups excreted more fecal BMG than did the control group on days 2 to 6, and the W/C group excreted more BMG than did the control group on days 2 to 4. Paired daily comparisons showed that the L-aspartic acid group excreted more BMG than did the W/C group on days 5 to 6. Overall fecal bilirubin excretion was significantly higher than control values in the L-aspartic acid (P < .001) and W/C (P = .0022) groups. The L-aspartic acid group excreted more fecal bilirubin than did the control group on days 4 to 6, and the W/C group excreted more bilirubin than did the control group on days 2 to 4. Fecal BP excretion resembled bilirubin excretion because the magnitudes of BDG and BMG fecal excretion were only 0.13% and 21% that of bilirubin, respectively. BDG, BMG, and bilirubin fecal excretion did not differ significantly between the L-aspartic acid and EHC groups. Dosages received among the treatment groups showed no significant differences in number of doses per day (P = .42), number of feedings per day (P = .74), total dosage volume per day (P = .80), or percentage of theoretical cumulative dose received (P = .99).
Follow-up information showed no significant differences in day 7 weight, number of day 6/7 weighed feedings, weight of day 6/7 milk and treatment, weighed day 6/7 milk, age at first formula feeding, breastfeeding cessation age, and mother’s rating of at least good (Table 2). There were no significant differences among the groups in meconium clearance, as determined with fecal zinc coproporphyrin measurements (P = .59), or in fecal urobilinoid levels (P = .43). There were no known adverse events or side effects. None of the infants demonstrated excessive weight loss (ie, >10%).
DISCUSSION
Our results showed that breastfed newborns given L-aspartic acid, EHC, or W/C during the first week had significantly lower transcutaneous bilirubin levels than did control subjects. We hypothesized that feeding -glucuronidase inhibitors (L-aspartic acid or EHC) would inhibit -glucuronidase present in the intestine. -Glucuronidase (-G) hydrolyzes glucuronic acid from bilirubin conjugates, converting BDG to BMG and BMG to bilirubin as illustrated.
If -glucuronidase is inhibited, then there should be relative preservation of conjugates on the left side of this equation. The fate of bilirubin is more complicated, however, because bilirubin is more easily absorbed from the intestine than are bilirubin conjugates30,31 and because intestinal bacteria can convert bilirubin and its conjugates to urobilinoids, a family of reduction-oxidation products.32 If bilirubin is not absorbed or converted to urobilinoids, then it is excreted in feces. The EHC group excreted significantly more fecal BDG and BMG than did the control group. The L-aspartic acid group also excreted significantly more BMG than did the control group. Interestingly, the W/C group also excreted more BDG and BMG than did the control group. Because W/C lacks -glucuronidase inhibitors, some other mechanism must underlie this observation. One possibility is increased binding of bilirubin conjugates by components of the W/C supplement in the intestinal lumen. Overall, however, fecal excretion of bilirubin glucuronides was minor, compared with total fecal bilirubin excretion (predominantly unconjugated). This underscores the importance of -glucuronidase, because little unconjugated bilirubin is excreted into bile. Bilirubin excretion was significantly higher in the L-aspartic acid group, compared with the control group, consistent with inhibition of -glucuronidase in the L-aspartic acid group.
Important events occur at the time of birth regarding bilirubin metabolism. Bilirubin glucuronides are present in fetal bile by 22 weeks of gestation and increase in concentration with the maturation of hepatic bilirubin glucuronosyltransferase.33 In the intestinal lumen, bilirubin glucuronides can undergo hydrolysis by -glucuronidase, producing bilirubin that is more easily reabsorbed into the circulation.30,31 In the fetus, this mechanism facilitates placental clearance of bilirubin. After birth, however, this enterohepatic bilirubin circulation delays clearance and prolongs jaundice. Although bilirubin glucuronosyltransferase does not plateau at adult levels until 14 weeks of age,34 the small but statistically significant gestational age differences among our groups cannot explain the findings in this study, because only the L-aspartic acid group had a mean gestational age greater than that of the control group.
An important consideration is any effect on breastfeeding. The AAP promotes breastfeeding as the best infant nutrition5,6 and recommends against supplements (eg, water, glucose-water solution, or formula) unless a medical indication exists.6 The AAP Clinical Practice Guideline regarding neonatal hyperbilirubinemia recommends against routine supplementation of nondehydrated breastfed infants with water or dextrose-water solution, noting that this does not prevent hyperbilirubinemia. It also lists exclusive breastfeeding as a major risk factor for the development of severe hyperbilirubinemia and exclusive bottle-feeding as a factor associated with decreased risk of significant jaundice.2 In the management of jaundiced breastfed infants, the AAP notes that temporary interruption of breastfeeding, with the substitution of formula, can reduce bilirubin levels; if supplemental fluid is needed, then the best fluid to use is a milk-based formula, because it inhibits the enterohepatic circulation of bilirubin and should help lower serum bilirubin levels. Our data showed that feeding of L-aspartic acid, EHC, or W/C in volumes of up to 30 mL per day had no deleterious effect on weight gain, milk intake per feeding, age at subsequent first supplementation, or duration of breastfeeding. Mothers approved the feedings, with 100% rating their study experience as good or excellent in the control, L-aspartic acid, and EHC groups. Estimated energy intake attributable to study nutrients was negligible (<50 J/day, <13.8 J/kg of birth weight per day). Our experience was similar to that of Nicoll et al,35 who showed that 38.6 mL of 5% dextrose per 1 kg of birth weight per day had no deleterious effect on breastfeeding, and our study used much less.
As judged by the age at first supplementation and the age at breastfeeding cessation, this study included many highly motivated, breastfeeding mothers. However, mothers could benefit from this approach independent of breastfeeding duration, because the approach was limited to the first week of life. The interventions were simple and well tolerated, as verified by the high ratings parents gave the study after completion. As indicated in Table 2, mothers rated this study very highly, and it is unlikely they would do so if these interventions were problematic. Therefore, we think that such interventions can be quite acceptable to both mothers and pediatricians, as part of a breastfeeding support/education program.
The 2004 AAP hyperbilirubinemia guidelines2 include a nomogram developed by Bhutani et al36 to classify serum bilirubin concentrations into 4 different risk zones. Seventeen of our subjects were in a risk zone above the low-risk zone within the first 48 hours after birth (6 in the control group, 5 in the L-aspartic acid group, 1 in the EHC group, and 5 in the W/C group). The decline times (in hours) from a risk zone above the low-risk zone within the first 48 hours to a low-risk zone after 48 hours were compared among the 4 groups for these 17 subjects. There were no significant differences in decline times between the 2 groups that received no -glucuronidase inhibitor (control: mean ± SD: 49.00 ± 34.01 hours; median: 31.00 hours; W/C: mean ± SD: 48.90 ± 5.70 hours; median: 49.58 hours; P = .41). There were also no significant differences in decline times between the 2 groups that received a -glucuronidase inhibitor (L-aspartic acid: mean ± SD: 25.23 ± 16.84 hours; median: 14.67 hours; EHC: mean ± SD: 23.58 ± 0 hours; median: 23.58 hours; P = 1.0). The combined inhibitor groups had a significantly shorter decline time than the combined no-inhibitor groups (inhibitor: mean ± SD: 24.96 ± 15.08 hours; median: 19.13 hours; no-inhibitor: mean ± SD: 48.95 ± 24.32 hours; median: 45.33 hours; P = .039, Wilcoxon test) (Fig 3). Only 1 subject in our entire study was in the highest (>95th percentile) risk zone (age: 22.7 hours). That subject was in the EHC group, and the risk decreased to the next-to-lowest risk zone (40–75th percentile) at 46.3 hours and to the lowest risk zone at 74.9 hours. It would be of interest to evaluate the effect of -glucuronidase inhibitors on larger numbers of infants in high-risk zones.
Although 64 infants were recruited into the study, when the infants were randomized into 4 groups each group became rather small. Nevertheless, our study showed significant differences clearly. Because this was a controlled, randomized, double-blind trial, we have confidence in its real clinical significance. However, future studies with larger sample sizes are warranted. Our treatment groups had peak bilirubin levels 25% to 30% lower than control values, and those that received the -glucuronidase inhibitor did decrease to lower risk zones faster than the groups that did not receive the -glucuronidase inhibitor. Whether this would be helpful for infants with higher bilirubin levels is not addressed with these data. However, if infants with bilirubin levels in the high-risk zone decreased their levels by 25% to 30%, this certainly could be helpful.
It was the hope of this study that breastfed infants could have the best of both worlds, ie, the advantages of exclusive breastfeeding plus the decreased risk of severe hyperbilirubinemia associated with exclusive formula feeding. The results show that significant reductions in transcutaneous bilirubin levels can be achieved, with no negative effect on breastfeeding, through administration of small daily amounts of selected key ingredients of currently available infant formula. In the L-aspartic acid and EHC groups, the effect probably results from -glucuronidase inhibition and increased fecal excretion of bilirubin. Lower transcutaneous bilirubin levels in the W/C group suggest a different mechanism. These results require confirmation with additional research involving larger numbers of subjects.
ACKNOWLEDGMENTS
This work was supported in part by grants from Mead Johnson Nutritionals, the University of Wisconsin Technology and Innovation Fund, and the University of Wisconsin Department of Pediatrics and Medical School.
Sincere thanks go to the following nurses: Mona Borgman, Sarah Goebel, Gynel Hagemann, Carmen McDonald, and Deb Wolff. Thanks also go to Deb Kundert and Pat Berra. We are greatly indebted to Dr Bruce Duemler, his co-workers, and the staff of the Monroe Hospital and Clinic (Monroe, WI), who embraced and supported this study enthusiastically, and to Dr John D. Bancroft. Thanks go to Scott Kerr, Respironics, for providing Bilicheck devices. This manuscript is dedicated to the memory of Dr Gerard B. Odell.
FOOTNOTES
Accepted Nov 23, 2004.
No conflict of interest declared.
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