Alan M. Kleinfeld

Fatty Acid Transport in Adipocytes Monitored by Imaging Intracellular Free Fatty Acid Levels

Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membranes role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFAi]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFAi] was measured by microinjecting cells with ADIFAB, a fluorescently labeled fatty acid-binding protein that is used to measure unbound FFA levels. We used ratio fluorescence microscopy of intracellular ADIFAB to image unbound FFA levels and determined the time course of [FFAi] in response to changing the extracellular unbound FFA concentration ([FFAo]). [FFAo] was clamped at defined levels using complexes of FFA and bovine serum albumin. We show that FFA influx is slow, requiring about 300 s to reach steady state (rate constant ∼ 0.02 s-1) and saturable (Ko ∼ 200 nm). Efflux is twice as fast as influx, for zero [FFAo], but decreases with increasing [FFAo]. Surprisingly, at steady state [FFAi] is 2–5-fold (average 2-fold) greater than [FFAo] and this [FFAi]/[FFAo] gradient is abolished by depleting cellular ATP. Our results indicate that FFA transport across adipocyte membranes is highly regulated, involving an ATP-driven pump and a mechanism for gating efflux that is sensitive to [FFAo]. These characteristics are well described by a membrane carrier model but are not consistent with FFA transport across the membranes lipid phase. We suggest that these characteristics are important in regulating circulating FFA levels by the adipocyte.

Stopped-flow kinetic analysis of long-chain fatty acid dissociation from bovine serum albumin

The kinetics of the interaction of long-chain fatty acids (referred to as fatty acids) with albumin is critical to understanding the role of albumin in fatty acid transport. In this study we have determined the kinetics of fatty acid dissociation from BSA and the BSA-related fatty acid probe BSA-HCA (BSA labelled with 7-hydroxycoumarin-4-acetic acid) by stopped-flow methods. Fatty acid-albumin complexes of a range of natural fatty acid types and albumin molecules (donors) were mixed with three fatty acid-binding acceptor proteins. Dissociation of fatty acids from the donor was monitored by either the time course of donor fluorescence/absorbance or the time course of acceptor fluorescence. The results of these measurements indicate that fatty acid dissociation from BSA as well as BSA-HCA is well described by a single exponential function over the entire range of fatty acid/albumin molar ratios used in these measurements, from 0.5:1 to 6:1. The observed rate constants (k(obs)) for the dissociation of each fatty acid type reveal little or no dependence on the initial fatty acid/albumin ratio. However, dissociation rates were dependent upon the type of fatty acid. In the case of native BSA with an initial fatty acid/BSA molar ratio of 3:1, the order of k(obs) values was stearic acid (1.5 s(-1)) < oleic acid < palmitic acid congruent with linoleic acid<arachidonic acid (8 s(-1)) at 37 degrees C. The corresponding values for BSA-HCA were about half the values for BSA. The results of this study show that the rate of fatty acid dissociation from native BSA is more than 10-fold faster than reported previously and that the off-rate constants for the five primary fatty acid-binding sites differ by less than a factor of 2. We conclude that for reported rates of fatty acid transport across cell membranes, dissociation of fatty acids from the fatty acid-BSA complexes used in the transport studies should not be rate-limiting.

Unbound free fatty acid concentrations are increased in cardiac ischemia

Monitoring increased plasma unbound free fatty acid (FFAu) concentrations has been proposed as a biomarker for myocardial ischemia. In the current study, 30 acute coronary syndrome (ACS) patients presenting in the emergency department, with chest pain within 12 h of onset, were clinically evaluated along with serial cardiac troponin I (cTnI) and FFAu measurements.Increased FFAu were found in 28 of 30 (93%) of ACS patients, ranging from 2.0 to 430 nM. For the nine ACS patients with myocardial infarction (MI), FFAu levels were increased at presentation for all (100%). In contrast, cTnI was increased in only 9 of 30 (30%) patients, mean 0.7 μg/L, and in only 2 of 9 (22%) MI patients, mean 1.3 μg/L. During the 24 h following admission, cTnI increased in all 9 MI patients. FFAu concentrations increased in every sample in which cTnI increased.Our findings suggest that FFAu is increased in ischemia regardless of the presence or absence of myocardial necrosis, as reflected by increased or normal cTnI, respectively.

Effects of Perinatal Hypoxia on Serum Unbound Free Fatty Acids and Lung Inflammatory Mediators

Cellular injury during tissue hypoxia is due, in part, to reactive intermediates released by activated leukocytes. We found that the inflammatory mediators tumor necrosis factor (TNF)-α, IL-6, and IL-1β are elevated in situ in lung macrophages on day 14 following exposure of rats to intermittent or chronic hypoxia from birth. Because inflammatory mediators can increase lipolysis in adipocytes, we also measured serum unbound free fatty acids (FFAu) – the biologically active compartment of FFA – in rat pups exposed to intermittent or chronic hypoxia. FFAu values were markedly elevated during the first 2 days of life in all rats, displaying an approximately 3-fold decrease from day 2 to day 3. Exposure to chronic hypoxia significantly increased FFAu levels measured on day 13. Since elevated serum FFAu are known to suppress leukocyte activation, we speculate that increased FFAu levels represent a mechanism for attenuating inflammation and tissue injury following sublethal hypoxia in the perinatal period, either physiologically in the immediate newborn period, or as a late response to ongoing hypoxic insult.

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.

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 500–2000u2009g; GA 23–34 weeks) using the fluorescent probe ADIFAB2 and enzymatic method. Results: The infants’ BW was 1266.2u2009±u2009440.7u2009g and GA 28.8u2009±u20093.1 weeks. TG levels were 77.4u2009±u200950u2009mg/dL, 140.2u2009±u2009188u2009mg/dL (pu2009<u2009.04 compared to levels during low dose IL infusion) and 135.6u2009±u2009118u2009mg/dL (pu2009<u2009.004), respectively during increased IL rates. FFAu levels were 17.7u2009±u200913u2009nM, 47.3u2009±u2009102.8u2009nM (pu2009=u2009.07) and 98u2009±u2009234u2009nM (pu2009=u2009.03). 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 500–2000u2009g; 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 13.6u2009±u20099.0u2009nM. 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.