Effect of reduced versus usual lipid emulsion dosing on bilirubin neurotoxicity and neurodevelopmental impairment in extremely preterm infants: study protocol for a randomized controlled trial.

BACKGROUND: Bilirubin neurotoxicity (BN) occurs in premature infants at lower total serum bilirubin levels than term infants and causes neurodevelopmental impairment. Usual dose lipid infusions in preterm infants may increase free fatty acids sufficiently to cause bilirubin displacement from albumin, increasing passage of unbound bilirubin (UB) into the brain leading to BN and neurodevelopmental impairment not reliably identifiable in infancy. These risks may be influenced by whether cycled or continuous phototherapy is used to control bilirubin levels. OBJECTIVE: To assess differences in wave V latency measured by brainstem auditory evoked responses (BAER) at 34-36 weeks gestational age in infants born ≤ 750 g or < 27 weeks' gestational age randomized to receive usual or reduced dose lipid emulsion (half of the usual dose) irrespective of whether cycled or continuous phototherapy is administered. METHODS: Pilot factorial randomized controlled trial (RCT) of lipid dosing (usual and reduced) with treatment groups balanced between cycled or continuous phototherapy assignment. Eligible infants are born at ≤ 750 g or < 27 weeks' gestational age enrolled in the NICHD Neonatal Research Network RCT of cycled or continuous phototherapy. Infants will randomize 1:1 to reduced or usual dose lipid assignment during the first 2 weeks after birth and stratified by phototherapy assignment. Free fatty acids and UB will be measured daily using a novel probe. BAER testing will be performed at 34-36 weeks postmenstrual age or prior to discharge. Blinded neurodevelopmental assessments will be performed at 22-26 months. Intention-to-treat analyses will be performed with generalized linear mixed models with lipid dose and phototherapy assignments as random effects covariates, and assessment for interactions. Bayesian analyses will be performed as a secondary analysis. DISCUSSION: Pragmatic trials are needed to evaluate whether lipid emulsion dosing modifies the effect of phototherapy on BN. This factorial design presents a unique opportunity to evaluate both therapies and their interaction. This study aims to address basic controversial questions about the relationships between lipid administration, free fatty acids, UB, and BN. Findings suggesting a reduced lipid dose can diminish the risk of BN would support the need for a large multicenter RCT of reduced versus usual lipid dosing. TRIAL REGISTRATION: Clinical Trials.gov, NCT04584983, Registered 14 October 2020, https://clinicaltrials.gov/ct2/show/NCT04584983 Protocol version: Version 3.2 (10/5/2022).

Unbound bilirubin levels in phototherapy-treated preterm infants receiving soy-based lipid emulsion.

BACKGROUND: Phototherapy is an effective treatment for neonatal jaundice. Treatment indication uses total serum bilirubin (TSB), although unbound bilirubin (Bf) more accurately predicts disability risk. The goals of this investigation were to examine the response of Bf and TSB to phototherapy in preterm infants, and we hypothesized that (i) TSB and Bf respond differently; (ii) the relationship between TSB and Bf is altered; and (iii) unexpected Bf elevations are found. METHODS: One hundred and seventeen preterm infants <2 kg at birth and receiving (IL) were enrolled; and measurements of TSB and Bf were obtained. TSB was measured by the diazo method and Bf with a fluorescent Bf sensor BL22P1B11-Rh. RESULTS: Initial mean (± SD) TSB and Bf levels (41.4 ± 6.9 h) were 8.0 ± 9.0 mg/dL and 16.9 ± 12.4 nmol/L (P < 0.05). The rates of rise (ROR) were 0.21 ± 0.10 mg/dL/h for TSB and 0.38 ± 0.33 nmol/L/h for Bf. Phototherapy reduced TSB from 8.0 ± 9.0 to 5.8 ± 9.4 mg/dL (P = 0.068) but Bf did not change (16.9 ± 12.4 to 14.1 ± 9.4 nmol/L P = n.s.). Bf levels were >11 nmol/L in 64, >17 nmol/L in 18, and >22 nmol/L in 7 infants. CONCLUSIONS: Bf and TSB responded differently. While TSB and Bf correlated well before phototherapy, they did not correlate during phototherapy. TSB showed a trend toward a reduction with treatment, Bf did not. While TSB ROR information is not helpful, ROR Bf data can be utilized to anticipate treatment. Potentially high Bf levels existed before and after phototherapy and the mean Bf level at phototherapy termination remained elevated in a significant proportion of 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.

Effect of Reduced Versus Usual Lipid Emulsion Dosing on Bilirubin Neurotoxicity and Neurodevelopmental Impairment in Extremely Preterm Infants: Study Protocol for a Randomized Controlled Trial.

Background : Bilirubin neurotoxicity ( BN ) occurs in premature infants at lower total serum bilirubin levels than term infants and causes neurodevelopmental impairment. Usual dose lipid infusions in preterm infants may increase free fatty acids sufficiently to cause bilirubin displacement from albumin, increasing passage of unbound bilirubin ( UB ) into the brain leading to BN and neurodevelopmental impairment not reliably identifiable in infancy. These risks may be influenced by whether cycled or continuous phototherapy is used to control bilirubin levels. Objective : To assess differences in wave V latency measured by brainstem auditory evoked responses ( BAER ) at 34-36 weeks gestational age in infants born ≤750 g or <27 weeks' gestational age randomized to receive usual or reduced dose lipid emulsion (half of the usual dose) irrespective of whether cycled or continuous phototherapy is administered. Methods : Pilot factorial randomized controlled trial ( RCT ) of lipid dosing (usual and reduced) with treatment groups balanced between cycled or continuous phototherapy assignment. Eligible infants are born at ≤750 g or <27 weeks' gestational age enrolled in the NICHD Neonatal Research Network RCT of cycled or continuous phototherapy. Infants will randomize 1:1 to reduced or usual dose lipid assignment during the first 2 weeks after birth and stratified by phototherapy assignment. Free fatty acids and UB will be measured daily using a novel probe. BAER testing will be performed at 34-36 weeks postmenstrual age or prior to discharge. Blinded neurodevelopmental assessments will be performed at 22-26 months. Intention-to-treat analyses will be performed with generalized linear mixed models with lipid dose and phototherapy assignments as random effects covariates, and assessment for interactions. Bayesian analyses will be performed as a secondary analysis. Discussion : Pragmatic trials are needed to evaluate whether lipid emulsion dosing modifies the effect of phototherapy on BN. This factorial design presents a unique opportunity to evaluate both therapies and their interaction. This study aims to address basic controversial questions about the relationships between lipid administration, free fatty acids, UB, and BN. Findings suggesting a reduced lipid dose can diminish the risk of BN would support the need for a large multicenter RCT of reduced versus usual lipid dosing. Trial Registration : Clinical Trials.gov, NCT04584983, Registered 14 October 2020, https://clinicaltrials.gov/ct2/show/NCT04584983 Protocol Version : Version 3.2 (10/5/2022).

Fatty acid (FFA) transport in cardiomyocytes revealed by imaging unbound FFA is mediated by an FFA pump modulated by the CD36 protein.

Free fatty acid (FFA) transport across the cardiomyocyte plasma membrane is essential to proper cardiac function, but the role of membrane proteins and FFA metabolism in FFA transport remains unclear. Metabolism is thought to maintain intracellular FFA at low levels, providing the driving force for FFA transport, but intracellular FFA levels have not been measured directly. We report the first measurements of the intracellular unbound FFA concentrations (FFA(i)) in cardiomyocytes. The fluorescent indicator of FFA, ADIFAB (acrylodan-labeled rat intestinal fatty acid-binding protein), was microinjected into isolated cardiomyocytes from wild type (WT) and FAT/CD36 null C57B1/6 mice. Quantitative imaging of ADIFAB fluorescence revealed the time courses of FFA influx and efflux. For WT mice, rate constants for efflux (∼0.02 s(-1)) were twice influx, and steady state FFA(i) were more than 3-fold larger than extracellular unbound FFA (FFA(o)). The concentration gradient and the initial rate of FFA influx saturated with increasing FFA(o). Similar characteristics were observed for oleate, palmitate, and arachidonate. FAT/CD36 null cells revealed similar characteristics, except that efflux was 2-3-fold slower than WT cells. Rate constants determined with intracellular ADIFAB were confirmed by measurements of intracellular pH. FFA uptake by suspensions of cardiomyocytes determined by monitoring FFA(o) using extracellular ADIFAB confirmed the influx rate constants determined from FFA(i) measurements and demonstrated that rates of FFA transport and etomoxir-sensitive metabolism are regulated independently. We conclude that FFA influx in cardiac myocytes is mediated by a membrane pump whose transport rate constants may be modulated by FAT/CD36.

Fluorescence sensor for the quantification of unbound bilirubin concentrations.

BACKGROUND: Hyperbilirubinemia in jaundiced neonates is routinely assessed by use of total serum bilirubin. However, the unbound or free form (B(f)), not total bilirubin, crosses the blood-brain barrier and can be neurotoxic. Although the peroxidase-mediated oxidation of bilirubin can be used to measure plasma concentrations of B(f), this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying B(f) in plasma. METHODS: Our method uses a mutated fatty acid binding protein labeled with the fluorescent molecule acrylodan (BL22P1B11), whose fluorescence is quenched upon binding bilirubin. Another configuration (BL22P1B11-Rh) was developed that uses BL22P1B11 together with the fluorophore rhodamine B, which responds by a change in the ratio of its fluorescence. RESULTS: The "B(f) probes" were calibrated with aqueous solutions of bilirubin and yielded similar bilirubin dissociation constants [K(d) = 16 (1.5) nmol/L]. We used the probes to determine B(f) concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent B(f) values in both systems, and the B(f) probe results were in agreement with the peroxidase assay. B(f) measurements revealed that bilirubin-HSA binding was well described by 2 sites with K(d) values of 15.4 (1) nmol/L and 748 (14) nmol/L. We measured B(f) concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%. CONCLUSIONS: The BL22P1B11-Rh probe provides accurate plasma sample B(f) concentrations with a single measurement, in 1 min with either a handheld B(f) meter or a laboratory fluorometer.

Neonatal hyperbilirubinemia and the role of unbound bilirubin.

BACKGROUND: Neonatal jaundice occurs in more than 80% of newborn infants. Although mild jaundice is physiologic and possibly neuroprotective, severe hyperbilirubinemia can lead to neurologic dysfunction and death. Hyperbilirubinemia is due to an imbalance between bilirubin production and the developing excretory capacity in the first days of life. Management utilizes total serum bilirubin (TSB) levels, although recent advances suggest a role for unbound bilirubin. GOALS: The goal of this review is to examine bilirubin biology, toxicology, and clinical effects, discuss preventive and therapeutic measures, describe neurodevelopmental consequences, and propose that, with the advent of new technology, unbound bilirubin is the optimal measurement for the management. METHODS: Comprehensive review on neonatal hyperbilirubinemia. RESULTS: Neonatal hyperbilirubinemia can be prevented by tin mesoporphyrin to limit heme oxygenase activity, a key enzyme in bilirubin production, or restricting bilirubin's absorption from the gastrointestinal tract. Treatment modalities include removing bilirubin from the body by exchange transfusion, binding to immunoglobulin, or converting it to a water-soluble isomer with phototherapy. While these approaches have evolved during the past decades, the diagnosis, intervention indications, and prognosis have consistently relied on TSB concentration despite its poor ability to predict an outcome. CONCLUSIONS: Total serum bilirubin is inadequate to optimize care of the term and preterm infant with hyperbilirubinemia. A rapid, accurate, and more effective indicator of bilirubin neurotoxicity is needed to manage jaundiced infants and for the universal screening of newborn infants. Future measurements of free bilirubin unattached to albumin will improve the management of neonatal hyperbilirubinemia.

Unbound bilirubin measurements in term and late-preterm infants.

BACKGROUND: Hyperbilirubinemia occurs in over 80% of newborns, and severe bilirubin toxicity can lead to neurological dysfunction and death. Unbound bilirubin (Bf) levels predict the risk of neurodevelopmental handicap, although total serum bilirubin (TSB) is used to manage care. OBJECTIVE: To measure Bf levels in healthy infants, its relationship to TSB, and its response to phototherapy. We hypothesize unexpectedly high Bf levels, poor correlation with TSB and unpredictable response to phototherapy. DESIGN/METHODS: Healthy infants were studied with simultaneous TSB and Bf measurements. The clinical data recorded included ethnicity, gender, birth weight, gestational age, and mode of delivery, Apgar scores, breast/formula feeds, and phototherapy. RESULTS: One hundred thirty-two infants (3248.9 ± 509.2g, GA 38.7 ± 1.4 weeks), at mean age of the initial sample of 28.5 ± 15.6 h, had a TSB of 7.9 ± 2.7 mg/dl, and a Bf of 5.2 ± 3.2 nM. The correlation between Bf and TSB was significant but not between Bf and TSB for TSB >12 mg/dl. Bf >11nm were in 22.7% and >17 nM in 3.8% of infants. Post-phototherapy TSB and Bf levels were similar to those before treatment. CONCLUSIONS: The relationship between TSB and Bf in healthy infants is complex, with the inability of one to predict the other's level in infants with elevated TSB. The mechanism of bilirubin-related neurotoxicity suggests that the management of jaundice in healthy infants requires Bf measurements. Management of jaundice with TSB may result in more infants exposed to phototherapy. However, unexpected elevations of Bf occur in an apparently healthy population.

Effects of lipid emulsions on unbound bilirubin and response to phototherapy in preterm infants.

OBJECTIVE: Bilirubin-induced neurotoxicity is mediated by the fraction of total serum bilirubin (TSB) not bound to albumin (Bf). Unbound free fatty acids (FFAu) generated from lipid emulsions compete with bilirubin for albumin binding, increasing Bf. Soy-based (IL) and soy-MCT-olive-fish oil-based (SMOF) lipid emulsions contain different fatty acids with distinct albumin binding affinities. IL increases Bf in preterm infants, but the effects of SMOF on Bf are not known. Our objective was to compare changes in TSB, Bf, FFAu, and response to phototherapy in preterm infants receiving SMOF and IL. We hypothesized that SMOF would be associated with lower Bf and better response to phototherapy than IL. METHODS: Very preterm and low birth weight infants (<1500 g, <32 weeks) were infused with IL (n = 20) or SMOF (n = 20) as prescribed by providers. Phototherapy was prescribed using the standard care practice. FFAu profiles and levels, TSB, and Bf were measured on 0, 1, 2, and 3 g/kg/day of lipid infusion and at the initiation and termination of phototherapy. TSB was analyzed in the clinical laboratory using the diazo technique. FFAu and Bf were measured using fluorescent probes. RESULTS: Escalating doses of IL and SMOF increased FFAu levels and Bf, but not TSB. Phototherapy did not significantly decrease Bf for infants receiving either lipid. IL-treated infants had higher levels of unbound linoleic acid, and SMOF-treated infants had higher unbound arachidonic, oleic, and docosahexaenoic acids. CONCLUSIONS: IL and SMOF both increase Bf similarly, and phototherapy does not significantly affect Bf for infants receiving them.

Plasma unbound free fatty acid profiles in premature infants before and after intralipid infusion.

Background: Unbound free fatty acids (FFAu) are the bioactive fraction of plasma free fatty acids (FFA). Most plasma FFA are bound to albumin. Only when FFA dissociate from albumin, do they become biologically active.Objective: To measure the first FFAu profiles in human infants and to measure these profiles before and during intravenous administration of the soybean lipid, intralipid (IL).Study design: The study population was 16 premature infants, from a parent study of 130 infants with birth weights 500-2000 g and gestational age 23-34 weeks. The infants chosen had plasma samples of ≥120 µL (volume needed for each FFAu profile measurement) in the first day of life. Infants received IL infusions starting in the second day of life at 1 g/kg/day, increasing by 1-g/kg/day daily up to 3 g/kg/day. FFAu profiles were determined during IL infusion when plasma was available. Profiles are the concentrations of the nine most abundant long-chain FFAu and were determined using novel fluorescent probes.Results: Before intralipid infusion unbound myristic acid was the dominant FFAu, as high as 78% of the total FFAu (sum of the 9 FFAu). In contrast, unbound linoleic acid was 0% in all infants. With increasing infusion of IL to 3 g/kg/day, unbound linoleic increased to 26% of the total FFAu, with unbound oleic, myristic, and linolenic acid the second, third and fourth most abundant. The average total FFAu concentration also increased from 4 nM before intralipid to 53 nM at 3 g/kg/day. During IL infusion the FFAu profiles approached the fatty acid composition of intralipid at 3 g/kg/day.Conclusions: This first study of FFAu profiles in neonates revealed that before IL infusion unbound linoleic acid was zero in all 16 infants and levels of myristic acid were exceptionally large, as much as 78% of the total FFAu profile. These results suggest important and previously unrecognized roles of lipid metabolism in early development. Zero unbound linoleic acid before IL infusion may help promote closure of the ductus arteriosus but after IL infusion, synthesis of arachidonic from linoleic acid may tend to promote patency. The high levels of unbound myristate may be needed for immediate neonatal energy needs.

Flip-flop is the rate-limiting step for transport of free fatty acids across lipid vesicle membranes.

The mechanism of transport of free fatty acids (FFA) across lipid bilayer membranes remains a subject of debate. The debate is whether the rate-limiting step for transport is flip-flop across the membrane or dissociation into the aqueous phase. Recently, a new method for assessing dissociation was described in which fluorescein phosphatidylethanolamine (FPE) introduced into the outer leaflet of lipid vesicles was used to monitor FFA dissociation. Transport of FFA into vesicles containing both FPE in the outer leaflet and pyranine trapped in the inside aqueous phase revealed identical rate constants for quenching of FPE and pyranine fluorescence. Because no difference was observed in the time for FFA binding to the outer surface and flip-flop across the bilayer, it was concluded that dissociation was slower than flip-flop. Here, we used FPE and BSA to assess dissociation of oleate from lipid vesicles. In separate pyranine- or ADIFAB-containing vesicles, we assessed flip-flop. We found that the FPE and BSA transfer methods yielded equivalent rate constants for dissociation, which were 3-10-fold faster than that of flip-flop. We found that in vesicles containing both FPE and pyranine, pyranine fluorescence cannot be separated from FPE fluorescence. The identical rate constants for FPE and pyranine observed with vesicles containing both fluorophores reflected the dominance (20-fold) of FPE fluorescence at pyranine excitation and emission wavelengths. Because the dissociation rate constants are 3-10 times faster than the rate constants for flip-flop, flip-flop must be the rate-limiting step for the transport of FFA across lipid vesicles.

Unbound bilirubin measurements by a novel probe in preterm infants.

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.

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

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.

Transport of 13C-oleate in adipocytes measured using multi imaging mass spectrometry.

The mechanism of long chain free fatty acid (FFA) transport across cell membranes is under active investigation. Here we describe the use of multi imaging mass spectrometry (MIMS) to monitor intracellular concentrations of FFA and provide new insight into FFA transport in cultured adipocytes. Cells were incubated with 13C-oleate:BSA and either dried directly or dried after washing with a medium deprived of 13C-oleate:BSA. Cells were analyzed with MIMS using a scanning primary Cs+ ion beam and 12C-, 13C-, 12C14N-, 13C14N-) (or 12C 15N-) were imaged simultaneously. From these quantitative images the values of the 13C/ 12C ratios were determined in the intracellular lipid droplets, in the cytoplasm and outside the 3T3F442A adipocytes. The results indicate that after incubation with 13C-oleate:BSA the droplet 13C/ 12C ratio was 15 +/- 6%. This value is about 14-fold higher than the 13C/ 12C terrestrial ratio (1.12%). After washing the 13C-oleate:BSA, the droplet 13C/ 12C ratios decreased to 1.6 +/- 0.1%, about 40% greater than the natural abundance. Results for washed cells indicate that relatively little FFA was esterified. The unwashed cell results, together with the value of the lipid water partition coefficient, reveal that intracellular unbound FFA (FFAu) concentrations were on average about 4.5-fold greater than the extracellular FFAu concentrations. These results are consistent with the possibility that FFA may be pumped into adipocytes against their electro-chemical potential. This work demonstrates that MIMS can be used to image and quantitate stable isotope labeled fatty acid in intracellular lipid droplets.

Usefulness of serum unbound free fatty acid levels to predict death early in patients with ST-segment elevation myocardial infarction (from the Thrombolysis In Myocardial Infarction [TIMI] II trial).

Circulating total free fatty acid (FFA) levels are elevated early in myocardial infarction (MI) and have been associated with an increase in mortality. We investigated the association of serum unbound FFA (FFAu) levels with mortality in patients presenting with ST-segment elevation MI in the Thrombolysis In Myocardial Infarction II trial. The Thrombolysis In Myocardial Infarction II trial enrolled patients within 4 hours of chest pain onset. The patients were treated with a recombinant tissue plasminogen activator within 1 hour of enrollment. The FFAu concentration was evaluated in serum samples from 1,834 patients obtained at baseline, before therapy. The FFAu level was an independent risk factor for death as early as at 1 day of hospitalization and continued to be an independent risk factor for the >3.8 years of follow-up. When adjusted for other cardiovascular risk factors, the FFAu levels in the fourth versus the first quartile remained an independent risk factor for death from MI (hazard ratio 5.0, 95% confidence interval 1.9 to 13.0), all cardiac death (hazard ratio 2.4, 95% confidence interval 1.3 to 4.4), and all-cause death (hazard ratio 1.9, 95% confidence interval 1.2 to 3.1). Women were twice as likely to be in the upper 2 FFAu quartiles and had approximately twice the rate of death as men. In conclusion, FFAu elevation is 1 of the earliest molecular biomarkers of mortality in patients with ST-segment elevation MI and was independent of other risk factors known to affect the outcomes after ST-segment elevation MI.

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 (Bf) and unbound free fatty acid (FFAu) 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, FFAu 376 ± 496 nM, and Bf 42 ± 30 nM. Of the 3 infants who died, 2 had TSB <5.9 mg/dl but FFAu >580 nM and Bf >75 nM. Multiple regression revealed a major effect on Bf levels due to FFAu, indicating that Intralipid elevated levels of FFAu and Bf. Indomethacin or ibuprofen reduced Bf levels, most likely by reducing FFAu levels through lipase inhibition. Because displacement of Bf by FFAu decouples Bf from TSB, phototherapy may not reduce the risk of bilirubin or FFAu toxicity in Intralipid-treated ELBW infants.

Effects of bilirubin on neutrophil responses in newborn infants.

BACKGROUND: Newborns are susceptible to inflammatory diseases due to defects in clearing activated immune cells from tissues. Therefore, mechanisms have likely evolved to protect neonates from leukocyte-mediated cytotoxicity. Bilirubin has antioxidant activity, and it is possible that it also exerts effects on cellular immune responses in jaundiced infants. OBJECTIVES: We hypothesize that bilirubin increases expression of antioxidant genes and decreases production of inflammatory proteins in neonatal neutrophils. METHODS: Neutrophils were isolated from umbilical cord blood, and from adults for comparison, and treated with bilirubin (10-300 µmol/l, equivalent to unbound bilirubin 3-40 nmol/l), in the presence or absence of lipopolysaccharide (LPS). Expression of genes for antioxidant enzymes [superoxide dismutase (SOD), heme-oxygenase-1 (HO-1)] and heme-dependent enzymes involved in inflammation [NADPH oxidase-1 (NOX-1), cyclooxygenase-2 (COX-2)] was measured by PCR. Inflammatory cytokines were measured by bead array analysis using flow cytometry. RESULTS: We found that LPS induced production of interleukin (IL)-8, IL-1ß, and macrophage inhibitory protein-1ß (MIP-1ß). Bilirubin increased basal production of IL-8 and IL-1ß, but downregulated LPS-induced generation of IL-8 and MIP-1ß. It also upregulated SOD and HO-1 gene expression. We observed an unexpected bilirubin-induced increase in gene expression of NOX-1 in LPS-activated cells, and of COX-2 in both resting and activated cells. CONCLUSIONS: These findings suggest that bilirubin suppresses inflammation and increases antioxidant enzyme generation in activated neonatal neutrophils. The unexpected increases in NOX-1 and COX-2 expression may represent an early response, with physiologic effects mitigated by increased antioxidant activity. Further studies will be needed to define levels of bilirubin that optimize its protective effects, while minimizing potential inflammatory toxicity.

B-type natriuretic peptide and serum unbound free fatty acid levels after contemporary percutaneous coronary intervention.

OBJECTIVES: To determine the frequency and timing of B-type natriuretic peptide (BNP) and unbound free fatty acid (FFAu) elevation after percutaneous coronary intervention (PCI). DESIGN AND METHODS: Blood samples were collected from 55 patients undergoing PCI within 1 hour prior to PCI, immediately after PCI, 6 hours and 18-24 hours after PCI, and were analyzed for BNP and FFAu. RESULTS: There was a trend toward a rise in BNP levels at 18-24 hours post-PCI (65 vs. 45 pg/ml; p = 0.056). FFAu levels rose immediately after PCI and returned to baseline by 6 hours postprocedure (2.0 nM pre-PCI, 6.4 nM immediately post-PCI, 1.9 nM 6 hours post-PCI, and 2.2 nM 18-24 hours post-PCI; p < 0.0001). BNP and FFAu levels were elevated post-PCI in 17% and 82% of cases. CONCLUSIONS: PCI using short inflation times and coronary stenting are associated with a trend toward increased BNP levels at 18-24 hours post-PCI and a transient significant rise in FFAu levels.

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump.

The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.