Main

During fasting, circulating FFA in plasma are derived from the breakdown of stored triacylglycerols in AT, and they represent the major energy source for most tissues(1). AT lipolysis is the major regulator of the body's supply of lipid energy, because it controls the release of FAs into the systemic circulation.

The rate of lipolysis has been measured in animals and humans by the systemic Ra of glycerol and PA using stable or radioactive isotope tracer methods(2–6). Using labeled glycerol, the release of FA is calculated to be three times theRa of glycerol(7); when infusing labeled PA the total flux of FFA is calculated by dividing the Ra of PA by the ratio of the plasma free PA concentration to total FFA(8). The latter calculation is based on the assumption that there is no discrimination among FA during lipolysis, thus the plasma concentration of individual FFA is a direct reflection of the AT FA pattern. This assumption is not supported by in vitro studies on lipolysis(9, 10), which show a different release of individual FAs. According to these studies HSL preferentially releases polyunsaturated FAs(9, 10) and FA with a shorter chain length(10) from AT triglycerides under conditions of stimulated lipolysis. No in vivo data are available to date on the comparison of the turnover rates of PA and LLA in humans. In this study we measured simultaneously the Ra of PA and LLA, and we tested the hypothesis that LLA is preferentially released during lipolysis in comparison with PA.

METHODS

Patients . Ra of LLA and PA was measured simultaneously in seven infants whose clinical characteristics are presented in Table 1. All patients were admitted to the intensive care unit of the Department of Paediatrics of the University of Padua for respiratory failure. The inclusion criteria for the study were: 1) age less than 1 y, 2) fasting for at least 12 h or no parenteral nutrition with lipids for at least 24 h, 3) stable hemodynamic condition for at least 6 h before and during the study, and 4) diagnosis of sepsis or sepsis syndrome according to the criteria of Bone(11). Infants were excluded from the study when they presented with signs of liver and renal failure, seizures, insufficient oxygenation (constant saturation <85% with normal cardiac anatomy), or if they received blood products during the study.

Table 1 Clinical characteristics of the infants studied
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Three patients were young infants and four were newborns(Table 1). Only patient 5 was not appropriate for gestational age at birth, and at the time of the study her weight was below the 3rd centile; two other infants (patients 3 and 6) had an appropriate weight at birth, but they were below the 3rd centile at the time of the study.

All the medications given to patients from the time of admission to the intensive care unit were recorded, and no changes in the administration of drugs occurred during the studies. All infants had variable degrees of multiorgan involvement and received mechanical ventilation, all but one(patient 4) were treated with dopamine (5μg·kg-1·min-1), and patient 5 also required dobutamine (6 μg·kg-1·min-1). All patients had arterial and central venous lines placed for clinical monitoring. The newborns were kept under a servo-controlled radiant warmer to maintain skin temperature at 36.5°C; the infants were studied in a room with ambient temperature of about 25°C and relative humidity of 40%. Body temperature was monitored every 2 h.

PRISM(12) and TISS scores(13) were calculated upon admission and daily afterward including the day of the study (Table 1).

Nutritional regimen. All patients received only i.v. glucose and electrolytes during the study period providing 20.9 ± 5.4 kcal·kg-1·d-1 (range 14-31). None of the patients received nutrients by oral route. Patients 2, 6, and 7 received parenteral nutrition with Intralipid® 20% (Pharmacia, Stockolm, Sweden) before they were enrolled in the study.

Study protocol. [U-13-C]PA (purity 99%) and [U-13-C]LLA (purity 97%) were purchased from Martek Biosciences (Martek Columbia, MD). Chemical and isotopic purity were confirmed by conventional GC-MS. [U-13-C]PA and [U-13-C]LLA bound to human albumin (Merieux, Pasteur, Lyon, France) were prepared for i.v. infusion as described previously(14). The albumin-bound tracers were infused by mean of a calibrated syringe pump (syringe pump STC 521, Terumo Corp., Rome Branch, Rome, Italia). The infusion rate was 1.0 ± 0.26μmol·kg-1·h-1 and 1.19 ± 0.23μmol·kg-1·h-1 for PA and LLA, respectively.

Before the start of the infusion of the labeled FA, a percutaneous needle AT biopsy was performed under local anesthesia as described elsewhere(15). We used the arterial line as the sampling source and the central venous line for tracer infusion. Blood samples for determinations of plasma enrichments of PA and LLA were obtained at 0, 1.50, 2.00, and 2.10 h after the start of the infusion in all patients. The samples were placed in tubes containing EDTA and immediately centrifuged at 1300× g, and plasma was stored in tubes containing pyrogallol as antioxidant at -20°C until analyzed.

Analytical methods. Plasma lipids were processed as previously described(16). In brief, plasma lipid classes including FFA were extracted according to Folch et al.(17) after the addition of nonanoic and pentadecanoic acid (Sigma Chemical Co., Milan, Italy) for the quantification of FFA. Lipid classes were separated by thin layer chromatography developed with heptane:diisopropyl ether:acetic acid (80:40:3 by vol) and visualized with 1,2-dichlorofluorescein by comparison with authentic standards.

Derivatization of the FAs was performed by adding 2 mL of 3 M dry HCl methanol. The quantification of FA methyl esters of the plasma lipid classes were performed by Hewlett Packard capillary GC (HP 5890 Hewlett Packard, Milan, Italy) equipped with a fused-silica column (Supelcovax 10, 60 m × 0.20-mm internal diameter, 0.25-μm film thickness; Supelchem SRL, Milan, Italy), a flame-ionization detector (280°C), and a split-splitless injector used in splitless mode. FAs were identified by comparing retention times with known standards (Nu Chek. Prep, Elysian, MN), and absolute plasma concentrations were calculated by HP-Chem-station software (Hewlett Packard, Milan, Italy) using nonanoic acid and pentadecanoic acid as internal standards. All reagents were analytical grade. Appropriate response factors were used for the calculation of the absolute plasma concentrations of all individual FAs which were expressed in millimoles or micromoles/L. Plasma FA from 6-24 carbon atoms were used for the calculation of the mol% values(mol/100 mol).

All measurements of the isotopic enrichments of plasma-free palmitate and linoleate were carried out on a Fisons MD 800 GC-MS quadrupole mass spectrometer (Fisons, Milan, Italy). The chemical ionization mode with isobutane as gas was utilized for analysis. The determination of the PA and LLA was performed during the same gas chromatographic run on a 30-m capillary column (Omegavax 250 30 m × 0.25-μm fused silica, internal diameter, 0.25 μm, Supelchem SRL, Milan, Italy). Selective ion monitoring of both FAs was carried out at m/z 271-m/z 287 for natural and[13C]PA, and m/z 295-m/z 313 for natural and[13C]LLA, respectively. Each sample was measured in duplicate.

FAs in AT were directly derivatized with 2 mL of 3 M dry HCl methanol. After being vigorously vortexed and placed at 90°C for 60 min, the samples were neutralized with K2CO3 6% and extracted with 200 μL of hexane. They were subsequently injected in the GC for the FA composition. Concentrations as well as isotopic enrichments of PA and LLA of the albumin solutions were measured for each individual patient by GC and GC-MS.

Calculations. The PA and LLA Ra was calculated by isotope dilution(18) according to the following equation, Q = i ×(Ei/(Ep - 1)) where i is the rate of tracer infusion (μmol·kg-1·h-1) andEi and Ep are the isotopic enrichments of the infusate and plasma PA and LLA.

Data are expressed as mean ± SD and comparisons were performed by an unpaired t test. p < 0.05 was considered significant.

Ethical considerations. The study protocol was approved by the Ethical Committee on Human studies at the Department of Paediatrics of the University of Padua and was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from the patient's parents.

Medical care of each infant was unaffected by the study and supervised by the attending physician who also designated the infant's sedation schedule. Fentanyl and benzodiazepine were used according to our clinical protocol. The s.c. needle AT microbiopsies were performed under local anesthesia with lidocaine 2% and soon after the dose of sedation was due(15). No variation of heart rate and blood pressure and no signs of discomfort were observed during the procedure.

RESULTS

Substrates. Mean plasma concentrations of total FFA, free PA, and free LLA are reported in Table 2. The intersubject variability of PA and LLA plasma concentrations (SD = 56.3 and 26.5, respectively) could be related to the severity of the disease or to treatments. However, the intrasubject variability was always below 10% when the three last points of the study were considered, which allowed us to calculate the turnover of PA and LLA with the steady state equation (data not shown).

Table 2 Total FFA, palmitic and linoleic acid plasma concentrations, and mol% values of the seven critically ill infants
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The infants' AT composition is reported in Table 3. The molar percentage of PA in AT was higher than that of LLA (p < 0.001); there was no correlation between free plasma PA concentration and the molar percentage PA in AT (r = 0.25, p = 0.57), whereas the plasma concentration of free LLA correlated significantly with its molar percentage value in AT (r = 0.86, p = 0.009).

Table 3 Adipose tissue composition and fatty acid turnover
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PA and LLA transport. The Ra of PA was significantly higher than the Ra of LLA in all the infants(p = 0.005) (Table 3). However, the ratio of theRa to the respective FA in AT, used as the indicator of the relative rate of lipolysis of both FA from AT, was higher for LLA than for PA(p = 0.02) (Table 3).

There was no correlation between the Ra of PA and AT composition (r = 0.1, p = 0.8), whereas theRa of LLA exhibited a highly significant correlation with the AT composition (r = 0.97, p = 0.0001).

DISCUSSION

Previous studies have calculated FFA kinetics under the assumption of an nonselective mobilization of FAs from AT and calculated the whole turnover of FFA based on the Ra of PA by dividing the Ra of PA by the ratio of the concentrations of palmitate to total FFA(5, 7, 8). In vitro data, however, do not support this method of calculation(9, 10). We investigated simultaneously the kinetics of whole body PA and LLA in critically ill infants with sepsis or sepsis syndrome, a condition with a high degree of lipolysis. The Ra of PA found in our study (5.73± 2.79 μmol·kg-1·min-1) is consistent with the findings of a previous study in newborn infants by Bougneres et al.(5) who found a similar value of 5.30± 0.75 μmol·kg-1min-1. The study by Wolfe et al.(8) in normal adult volunteers (6.7± 1.35 μmol·kg-1·min-1) and in a mixed population of adults and children with severe burn injury (14.5 ± 4.71μmol·kg-1·min-1) reports higher figures of total FFA turnover, but the contribution of the Ra of PA to the total Ra of FFA cannot be calculated from the data given in the article.

Plasma concentrations of total FFA and free PA in our infants were within the normal range, suggesting that even at this high rate of substrate release,i.e. lipolysis, a significant substrate cycling (resynthesis of triglycerides) must have occurred(8).

We measured simultaneously the Ra of LLA and found a mean value of 1.34 μmol/kg/min, which is 23.5% of the mean Ra of PA. We calculated the ratio of the Ra of both FAs in relation to the molar percentages of the respective FA in AT. These ratios can be used as indicators of the lipolysis in relation to the composition of the AT. The higher ratio of LLA than that of PA (p = 0.02) indicates that LLA is preferentially released from AT during stress in critically ill infants.

This is the major finding of our study and it has methodologic as well as physiologic consequences. From a methodologic point of view our data show that the Ra of FFA cannot be calculated assuming that PA is representative of all FA. The preferential release of LLA could be even greater after the neonatal period when, due to dietary influences, LLA in AT increases and PA decreases(19, 20). In vitro studies support our finding of a preferential release of LLA and show that PA is 10% less easily mobilized from AT than total FA(10). The use of PA as tracer could lead to 2-3-fold underestimation of the Ra of highly unsaturated FAs, such as eicosapentaenoic acid (C20:5ω3) arachidonic (C20:4ω6), or docosahexaenoic acid (C22:6ω3).

From a physiologic point a selective mobilization of certain FA probably affects the AT FA composition and the qualitative FA supply to tissues and organs in situations of negative energy balance. We did not find a correlation between plasma PA concentration and either Ra or PA molar percentage in AT, whereas the Ra and plasma concentration of LLA were highly correlated with the respective AT composition. This could suggest again a different mobilization of the two FAs and a lower“retention” in fat cells of LLA. LLA, as all the polyunsaturated FAs, is a polar FA. The hydrolysis of triglycerides, the rate-limiting step in FAs mobilization, is catalyzed by the HSL and occurs at the interface of lipid droplets containing triglycerides and the aqueous cytoplasm containing the lipase. It has been suggested that the more polar triglycerides, which are the molecules richer in polar FA, are preferentially located at the interface and thus more accessible to the HSL(10). Recently it has also been suggested that the HSL exhibits a preference for certain triglycerides only under stimulated conditions (with isoproterenol), but that this effect is not present under basal conditions(21). Six infants in our study received a constant infusion of adrenergic drugs(dopamine and dobutamine), which could have increased AT lipolysis via stimulation of the β1 and β2 adrenoceptors(22). Future studies should answer the question as to whether the selective release of FAs in AT is present under“basal” conditions or whether it becomes more prominent during conditions of “high stimulation” such as in trauma and sepsis or with the use of β adrenergic drugs. The infants also received a constant i.v. infusion of 3 μg·kg-1·min-1 fentanyl. At this dosage fentanyl is reported to reduce the stress response in adult surgical patients(23) and in infants after cardiac surgery(24); however, no data are available on the effect on the Ra of plasma FFA.

Whether or not the selective release of polyunsaturated FAs (found in thein vitro studies) and of LLA (found in our in vivo study) under conditions of highly stimulated lipolysis recruits essential FAs for cell repair, for eicosanoid synthesis, and for the endogenous synthesis of long chain polyunsaturated FAs is an attractive hypothesis. We have recently shown that even small preterm infants are capable of synthesizing arachidonic and docosahexaenoic acid from LLA and linolenic acid(25), and whether synthesis of arachidonic and docosahexaenoic acid is altered in critically ill infants is an interesting question that needs to be addressed.

Cerra et al.(26) have undertaken a study of changes in the FA profile in serious trauma of closed head injury 2 d after injury. The FA profile showed a general lowering of ω6 essential FAs and an enhancement of the shorter chain saturated and monounsaturated acids. The same finding was reported in animal models with sepsis and in surgical patients(27, 28).

In conclusion, our in vivo study, in critically ill infants with a low energy intake, confirms the in vitro observations that theRa of LLA is higher than expected from the FA composition of AT. We speculate that the contribution of LLA to energy metabolism could be larger than what is normally assumed from the FA composition of AT and of plasma FFA.