Andrew Henry Huber

Unbound Free Fatty Acids from Preterm Infants Treated with Intralipid Decouples Unbound from Total Bilirubin Potentially Making Phototherapy Ineffective

Extremely low birth weight (ELBW; <1,000 g) infants have poor outcomes, often compromised by bilirubin neurotoxicity. We measured unbound bilirubin (B_f) and unbound free fatty acid (FFA_u) levels in 5 ELBW infants in a trial examining the effects of pharmacologic ductal closure on infants treated with Intralipid infusion (3 g/kg/day). The levels for all infants (mean ± SD) were: total serum bilirubin (TSB) 4.6 ± 1.7 mg/dl, FFA_u 376 ± 496 n<smlcap>M</smlcap>, and B_f 42 ± 30 n<smlcap>M</smlcap>. Of the 3 infants who died, 2 had TSB <5.9 mg/dl but FFA_u >580 n<smlcap>M</smlcap> and B_f >75 n<smlcap>M</smlcap>. Multiple regression revealed a major effect on B_f levels due to FFA_u, indicating that Intralipid elevated levels of FFA_u and B_f. Indomethacin or ibuprofen reduced B_f levels, most likely by reducing FFA_u levels through lipase inhibition. Because displacement of B_f by FFA_u decouples B_f from TSB, phototherapy may not reduce the risk of bilirubin or FFA_u toxicity in Intralipid-treated ELBW infants.

Unbound free fatty acid profiles in human plasma and the unexpected absence of unbound palmitoleate

We determined for the first time the profiles of the nine most abundant unbound FFAs (FFAus) in human plasma. Profiles were determined for a standard reference plasma of pooled healthy adults for which the Lipid MAPSMAPS Consortium had determined the total FFA profiles. Measurements were performed by using 20 different acrylodan-labeled fatty acid binding protein mutants (probes), which have complementary specificities for the nine FFAs that comprise more than 96% of long-chain plasma FFA. The acrylodan fluorescence emission for each probe changes upon binding a FFAu. The plasma concentrations of each of the nine FFAus were determined by combining the measured fluorescence ratios of the 20 probes. The total molar FFAu concentration accounted for <10−5 of the total FFA concentration, and the mole fractions of the FFAu profiles were substantially different than the total FFA profiles. Myristic acid, for example, comprises 22% of the unbound versus 2.8% of the total. The most surprising difference is our finding of zero unbound cis-9-palmitoleic acid (POA), whereas the total POA was 7.2%. An unidentified plasma component appears to specifically prevent the release of POA. FFAus are the physiologically active FFAs, and plasma FFAu profiles may provide novel information about human health.

Effects of Soybean Lipid Infusion on Unbound Free Fatty Acids and Unbound Bilirubin in Preterm Infants

Objective To assess the effects of a soybean lipid emulsion infusions on levels of unbound (free) bilirubin (Bf) and unbound free fatty acids (FFAu) as well as changes in Bf and total serum bilirubin (TSB) during phototherapy in infants born preterm. Study design Ninety‐seven infants born preterm (birth weight: 500‐2000 g; gestational age: 23‐34 weeks) were enrolled to investigate the effect of 0, 1, 2, and 3 g/kg/d of intralipid infusion on Bf and FFAu. Pre‐ and postphototherapy TSB, FFAu, and Bf also were analyzed in 91 infants to assess the effects of phototherapy. FFAu levels were measured with the fluorescent probe ADIFAB2 and Bf by the fluorescent Bf sensor BL22P1B11‐Rh during intralipid infusion and at start and end of phototherapy. TSB and plasma albumin were measured by the diazo and bromcresol green techniques, respectively. Bilirubin‐albumin dissociation constants were calculated based on Bf and plasma albumin. Results Bf and FFAu increased with increasing intralipid dosage across all gestational ages. TSB and Bf were correlated significantly when infants received 0 or 1 g/kg/d of intralipid but not at greater doses of intralipid (2 and 3 g/kg/d). Although phototherapy effectively reduced both TSB and Bf in the total phototherapy group (by 32% and 12%, respectively), it reduced TSB, but not Bf, in infants less than 28 weeks of gestation. Conclusions Increasing intralipid doses result in increasing FFAu levels, which are associated with increased Bf independent of TSB. In infants born extremely preterm (<28 weeks of gestation), phototherapy effectively reduces TSB but not Bf.

Effects of soybean lipid infusion on triglyceride and unbound free fatty acid levels in preterm infants

Abstract Objective: To determine the plasma triglyceride (TG) and unbound free fatty acid (FFAu) levels in infants treated with increasing dosages of soybean lipid, intralipid (IL), infusion. Study design: TG and FFAu levels were measured in 78 preterm infants (BW 500–2000 g; GA 23–34 weeks) using the fluorescent probe ADIFAB2 and enzymatic method. Results: The infants’ BW was 1266.2 ± 440.7 g and GA 28.8 ± 3.1 weeks. TG levels were 77.4 ± 50 mg/dL, 140.2 ± 188 mg/dL (p < .04 compared to levels during low dose IL infusion) and 135.6 ± 118 mg/dL (p < .004), respectively during increased IL rates. FFAu levels were 17.7 ± 13 nM, 47.3 ± 102.8 nM (p = .07) and 98 ± 234 nM (p = .03). TG levels correlated with IL dose, the rate of IL administration, and FFAu levels. TG and FFAu levels were higher in infants below 28 weeks’ gestation Conclusions: Increasing dosage of IL is associated with increasing levels of TG and FFAu, especially in infants below 29 weeks of gestation. The increased level of FFAu suggests inefficient cellular utilization.

Unbound bilirubin measurements by a novel probe in preterm infants

Abstract Background: Hyperbilirubinemia occurs in over 80% of newborns and severe bilirubin toxicity can lead to neurological dysfunction and death, especially in preterm infants. Currently, the risk of bilirubin toxicity is assessed by measuring the levels of total serum bilirubin (TSB), which are used to direct treatments including immunoglobulin administration, phototherapy, and exchange transfusion. However, free, unbound bilirubin levels (Bf) predict the risk of bilirubin neurotoxicity more accurately than TSB. Objective: To examine Bf levels in preterm infants and determine the frequency with which they exceed reported neurotoxic thresholds. Methods: One hundred thirty preterm infants (BW 500–2000 g; GA 23–34 weeks) were enrolled and Bf levels measured during the first week of life by the fluorescent Bf sensor BL22P1B11-Rh. TSB and plasma albumin were measured by standard techniques. Bilirubin-albumin dissociation constants (Kd) were calculated based on Bf and plasma albumin. Results: Five hundred eighty samples were measured during the first week of life, with an overall mean Bf of 13.6 ± 9.0 nM. A substantial number of measurements exceeded potential toxic thresholds levels as reported in the literature. The correlation between Bf and TSB was statistically significant (r2 0.17), but this weak relationship was lost at high Bf levels. Infants <28-week gestations had more hearing screening failures than infants ≥28-week gestation. Conclusions: Unbound (free) bilirubin values are extremely variable during the first week of life in preterm infants. A significant proportion of these values exceeded reported neurotoxic thresholds.