1Institute of Diabetes "Gerhardt Katsch" Karlsburg, D-17495 Karlsburg; 2Department of Clinical Pharmacology of the Peter Holtz Center of Pharmacology and Experimental Therapeutics, and 3Hospital Pharmacy, University of Greifswald, D-17487 Greifswald; and 4Department of Biochemistry and Physiology of Nutrition, German Institute of Human Nutrition, D-14558 Bergholz-Rehbrücke, Germany
Submitted 18 July 2002 ; accepted in final form 17 February 2003
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ABSTRACT |
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stable isotopes; fatty acids; leucine metabolism; urea production
To study the acute effects of NEFA on fibrinogen synthesis in fasting healthy volunteers, the fractional synthesis rate of fibrinogen, leucine kinetics, and the endogenous formation of urea during lipid infusion (Lipofundin) and administration of heparin were measured with stable isotope dilution techniques for 8 h.
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RESEARCH DESIGN AND METHODS |
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Study protocol. The interactions between NEFA metabolism, amino acid catabolism, and fibrinogen synthesis were investigated after experimental elevation of blood NEFA and TG concentrations by lipid infusion (Lipofundin) and administration of heparin compared with a saline placebo infusion and a sham subcutaneous injection. The washout period between control and test treatments was 14 days. The subjects were randomly allocated to the treatment sequence (block randomization). Two days before the respective metabolic study, they consumed a standard diet (2,925 kcal) consisting of 115 g protein, 95 g fat, and 380 g carbohydrates, which was eaten in four meals at 0900, 0100, 0400, and 0700. This caloric intake is recommended by the German Society of Nutrition (reference values for nutrient intake of the German Nutrition Society, Umschau/Braus, 1st ed., Frankfurt am Main 2000; ISBN 3-8295-7114-3) for subjects with a mean physical activity level between 1.6 and 1.8 (e.g., students). After this run-in period, the subjects were admitted to the research unit for the metabolic study at 7 AM after overnight fasting. Then, cannulas were placed in cubital veins of both forearms for infusion and collections of blood samples to measure baseline values 30 and 15 min and immediately before start of the infusions at 8 AM. The first indirect calorimetry and breath sampling tests were done between 30 and 10 min before the infusion. The arterialized blood samples were drawn after the forearm was heated with a heating pad. Bed rest was advised until 4 PM. All procedures were performed in the supine position.
To elevate blood NEFA concentrations, after a 10-ml prime infusion of Lipofundin (MCT 10%, Braun Melsungen, Melsungen, Germany), 60 ml of Lipofundin/h were continuously infused between 8 AM and 4 PM. Heparin (5,000 IU, Braun Melsungen) was injected subcutaneously at 8 AM and 12 noon. Noncarbonated mineral water (≤1.5 liters) could be drunk 2 h after start of the infusion.
To study protein, fibrinogen, and urea turnover, NaH13CO3, [1-13C]leucine, and [15N2]urea were administered in the following manner. NaH13CO3 (176 µmol dissolved in 10 ml of saline; Cambridge Isotope Laboratories, Andover, MA) and 454 µmol of [1-13C]leucine (dissolved in 20 ml of saline; Cambridge Isotope Laboratories) were injected within 2 min at 8 AM, followed by continuous infusion of 0.151 µmol·kg-1·min-1 of [1-13C]leucine for 8 h. At 12 noon, 13.5 mmol of [15N2]urea dissolved in 10 ml of saline were injected (Cambridge Isotope Laboratories) within 2 min and followed by a continuous infusion of 0.3 µmol·kg-1·min-1 of [15N2]urea for 4 h.
The tracers were administered with precision infusion pumps (Perfusor Secura FT, Braun Melsungen), and Lipofundin was infused with a roller pump (Infusomat, Braun Melsungen). All solutions were manufactured by the university hospital pharmacy according to the regulations of the German Medicines Act.
Blood samples for -[13C]ketoisocaproate ([13C]KIC) and fibrinogen analysis were taken between 0130 and 0400 in intervals of 30 min, and for urea analysis between 0200 and 0400 in intervals of 15 min. Total carbon dioxide production (
CO2) and oxygen consumption (
O2) rates were monitored with an indirect calorimeter (Datex Delta Trac, Metabolic Monitor, Instrumentarium, Helsinki, Finland) between 0130 and 0400 in intervals of 30 min. Breath was collected in rubber bags from which samples were immediately transferred into evacuated 15-ml glass tubes and stored at room temperature until analysis.
Analytical procedures. Plasma glucose was measured with the Beckman Analyzer 2 (Fullerton, CA). Hematocrit was determined with a micro method (Micros 60, ABS Diagnostics, Montpellier, France) and TG with an automated photometric method (COBAS, Cobas Mira, Hoffmann-La Roche, Basel, Switzerland). NEFA were analyzed with a commercial kit (NEFA C, Wako, Osaka, Japan). Plasma immunoreactive insulin, human C-peptide, and glucagon were assayed by RIA (Linco Research, St. Charles, MO). Plasma urea was analyzed according to the urease-glutamate dehydrogenase reaction (Hitachi Automatic Analyzer 911, Boehringer Mannheim, Germany). Fibrinogen concentration was measured photometrically after coagulation (BCT Dade Behring, Schwalbach, Germany) (10). Isolation of fibrinogen from plasma was performed with the -alanine method (27). Low-molecular-weight substances and proteins were removed by dialysis (50 kDa cutoff). The purity of the intact fibrinogen and of the
-chain (67 kDa),
-chain (56 kDa), and
-chain (47 kDa) produced by reductive cleavage was checked by SDS-PAGE and Coomassie brilliant blue staining. Five hundred microliters of the isolated fibrinogen solution (
23 mg protein/ml) were precipitated by 2 ml of ice-cold methanol in 16 x 100-mm tubes with polytetrafluoroethylene-lined caps. After cooling on ice for 30 min and centrifugation (250 g, 4°C, 10 min, RT 6000 D, Sorvall, Bad Homburg, Germany), the precipitate was dried under a gentle stream of nitrogen at 60°C (Vapotherm, Labor Technik Barkey, Leopoldshöhe, Germany). The residue was hydrolyzed in 2 ml of 6 M hydrochloric acid at 110°C for 24 h. The amino acids obtained from the hydrolyzed fibrinogen were purified by cation exchange chromatography (40).
The enrichment of fibrinogen-bound [13C]leucine was determined after derivatization to its N-pivaloyl-i-propyl ester by the isotope ratio mass spectrometer Delta S-IRMS (Finnigan, Bremen, Germany) coupled with the gas chromatograph HP 5890 (Hewlett-Packard, Waldbronn, Germany) via a combustion interface (3739). Briefly, the hydrolysate was dried and dissolved for derivatization in thionyl chloride/i-propanol solution and boiled at 100°C. The propylated product was dried and then dissolved in pyridine. After addition of pivaloyl chloride, the amino acids were acylated, and methylene chloride was added after cooling. Then the mixture was passed through a silica gel column, and the filtrate was dried under a gentle nitrogen stream. The residue was dissolved in ethyl acetate, 0.5 µl of which was injected (splitless, autosampler CTC A200S; CTC Analytics, Zwingen, Switzerland) into the chromatograph equipped with a capillary column (Ultra 2, 50 m length, 0.32 mm ID; Hewlett-Packard; carrier gas 1 ml/min helium). The injector temperature was 280°C. The column was heated with a gradient program as follows: 70°C for 1 min; 3°C/min increase to 160°C, which was maintained for 4 min; 10°C/min increase to 300°C, which was maintained for 2 min. The signal of the mass-to-charge ratio (m/z) 44 ion current that represented the leucine N-pivaloyl-i-propyl esters of an amino acid standard mixture or of the fibrinogen hydrolysate appeared after 36 min. Data processing was performed with the software ISODAT (Finnigan, Bremen, Germany). The 13C abundance was assessed relative to the international standard (PDB Belemnite carbonate), and 13C enrichment was expressed in atom percent (AP) and atom percent excess (APE) above baseline, as described recently (37). The 13C enrichment of leucine was corrected by the extra carbon that had been introduced during derivatization (37).
[13C]KIC enrichment was measured after derivatization of the analyte to the quinoxalinol-N-methyl-N-(tert-butyldimethylsilyl) derivative (15, 35) with the mass spectrometer SSQ 710 (Finnigan) coupled with the gas chromatograph Varian 3400 (Varian Chromatography Systems, Walnut Creek, CA) equipped with a DB-5 capillary column (30 m x 0.25 mm, 0.25 µm ID; J&W Scientific; Folsom, CA) to heat the sample (1 µl, splitless injection) from 100°Cto280°C with a rate of 30°C/min. Natural and [13C]KIC signals appeared after 6.9 min and were monitored at m/z 259 and 260 by selected ion monitoring. Tracer/tracee ratios were calculated from m+1 and m+0 area ratios. Graded mixtures of [13C]KIC and unlabeled KIC corrected for baseline were used for calibration (41).
13C enrichment in breath samples was analyzed by IRMS (Breath, Finnigan). CO2 was separated from N2 and O2 by use of a HayeSep-D packed steel column.
Urea was analyzed as described recently (16). Briefly, 0.5 ml of plasma was deproteinized with sulfosalicylic acid (15%) and subjected to cation exchange chromatography (Dowex 50 WX8, 200400 mesh; Serva, Heidelberg, Germany). The urea-containing fraction was eluted with 2 M ammonium hydroxide and dried in a nitrogen atmosphere. Then the samples were derivatized at 100°C with N,O-bis-trimethylsilyltrifluoroacetamide (Merck, Darmstadt, Germany) to the trimethylsilyl derivative. Fragment ions m/z 189, 190, and 191 (M-15) were monitored with the mass spectrometer SSQ 710 (Finnigan, San Jose, CA) with a DB 5-ms capillary column to estimate the [15N2]urea abundance. The mole percent excess (MPE) enrichment was calculated with established formulas (57).
Biometric evaluation. Leucine oxidation (Leuox) was calculated by the equation
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Leucine rate of appearance (LeuRa) was assessed using the mean isotope steady-state enrichment values after a significant slope during the last 2.5 h of the tracer protocol was excluded by linear regression analysis.
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Therefore, NOLD was calculated as
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The fractional synthesis rate (FSR) of fibrinogen was calculated by dividing the regression slope of fibrinogen isotope enrichment from 330 min to 480 min of leucine tracer infusion by the plasma plateau enrichment of [13C]KIC according to the precursor/product relationship
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The steady-state MPE values of plasma urea (all values between 0245 and 0400) were taken to calculate urea production in accordance with Wolfe (57)
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Statistical evaluation. Data are given as means ± SD. Differences between samples were evaluated by paired Student's t-test, with P < 0.05 as the level of significance.
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RESULTS |
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Leucine metabolism. Infusion of [1-13C]leucine for 5.5 h in subjects not treated with NEFA (controls) labeled the plasma KIC with 6.32 ± 0.63 MPE. Longer infusion and administration of Lipofundin and heparin were without additional effect (Fig. 3). 13CO2 breath enrichment was nearly identical in controls and lipid/heparin-treated subjects (after 5.5 h: 0.0151 ± 0.0018 vs. 0.0151 ± 0.0022 APE; after 8 h: 0.0157 ± 0.0016 vs. 0.0143 ± 0.0020 APE). Both [13C]KIC labeling and 13CO2 breath enrichment indicated comparable labeling of the intracellular leucine and the bicarbonate pool in both groups at steady state. This is considered to be a basic precondition for proper estimation of leucine (protein) metabolism and fibrinogen synthesis. LeuRa from endogenous protein breakdown, Leuox, and NOLD via protein synthesis were not influenced either by the study conditions or the administration of Lipofundin plus heparin, as shown in Fig. 3.
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Plasma fibrinogen and fibrinogen FSR. Plasma fibrinogen concentration was constant during the metabolic study and was not influenced by lipid and heparin (Fig. 4). Enrichment of fibrinogen with [1-13C]leucine showed a strong linear increase between 5.5 and 8 h of the tracer infusion in both controls and subjects with concomitant lipid and heparin administration. The slope of the regression line representing the change of fibrinogen-bound leucine enrichment vs. time was consistently lower in the lipid-heparin group than in the control group (P < 0.05). Therefore, the fibrinogen FSR was significantly lower in treated than in control subjects (18.44 ± 1.48 vs. 21.84 ± 1.37%, P < 0.05).
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Plasma urea and endogenous production. Short-term elevation of TG and NEFA did not change endogenous urea production significantly (Fig. 5). Plasma urea, [15N2]urea, and ureaRa were not influenced by the study conditions and the administration of Lipofundin and heparin.
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DISCUSSION |
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Infusion of lipid (Lipofundin) combined with heparin increased steady-state plasma NEFA and TG concentrations about threefold above the fasting levels. The elevation of TG in our study was higher than the peak levels observed in young healthy male subjects with low fasting TG levels after ingestion of 58 g of saturated fat, but it was lower than in subjects with high fasting TG (52). However, the NEFA concentrations in our study were markedly (three times) higher than those recently reported from obese nondiabetic female adolescents (1617 yr) in the postabsorptive state (2).
The 8-h lipid infusion and administration of heparin were without significant influence on leucine metabolism, overall protein catabolism, plasma insulin, and glucagon as characterized by LeuRa, Leuox, NOLD, endogenous urea production, data of gas exchange, and EE. These unchanged metabolic and hormonal conditions were appropriate factors for the investigation of the effect of elevated NEFA and TG levels on fibrinogen synthesis.
The expected changes in O2 and
CO2 after a high-caloric input occurred in our study at the end of the lipid loading and did not influence the linearity of the [13C]leucine-related labeling of fibrinogen, which was monitored during the final 2.5 h. On the other hand, NEFA for VLDL synthesis seems to be available in the liver quite early, because oral administration of long-chain TG was associated with transfer of NEFA from the plasma into the VLDL pool within 12 h (4).
The extent of lipid loading in our study has not changed the proportion of carbohydrates and lipids utilized for EE during the whole observation period, as indicated by constant respiratory quotient. The late reduction in glucose oxidation was not surprising, because, unlike the data of Roden et al. (49), our data were not obtained under hyperinsulinemic clamp conditions. In their investigation, the reduction of glucose oxidation occurred after 90120 min, and storage of glucose 6-phosphate in human calf muscle occurred after 180 min (48). Even after extreme loading with Intralipid and nine-times-elevated NEFA, as reported by Yki-Järvinen et al. (58), the switch to preferential consumption of lipids occurred faster and was associated with significantly reduced glucose oxidation after 2.5 h. However, this was observed under the conditions of euglycemic hyperinsulinemia (a metabolic clamp). Under the condition of nonclamped insulin and step-wise elevation of NEFA by infusion of increasing Lipofundin doses, Stingl et al. (53) observed late reduction in glycogenolysis after 400 min and an insulin increase in the last 3 h of the 9-h experiment.
The values of fibrinogen synthesis rates and protein (leucine) kinetics in our study were in agreement with data from other studies (13, 17, 54, 55). The amino acid catabolism was not influenced by experimental NEFA elevation, because formation of urea and leucine oxidation remained unchanged during the 8 h of lipid infusion. The results on urea production rates were comparable to the values obtained in healthy volunteers with use of an identical isotope dilution technique (34).
In summary, under comparable metabolic and hormonal conditions, in nine of the ten paired experiments, elevation of NEFA was significantly associated with lower fibrinogen synthesis rates, as shown in Fig. 4. This observation was contrary to our hypothesis, which was derived from data in the literature on the effect of chronic NEFA elevation on fibrinogen synthesis. As recently shown, NEFA elevation in obese insulin-resistant hyperinsulinemic young postpubertal females was associated with increased rates of fibrinogen synthesis (2).
Our study was performed in lean healthy subjects who were not resistant to insulin. Insulin was significantly decreased during fasting of the subjects, and blood glucose remained unchanged. After lipid infusion, insulin (including C-peptide) and glucagon were not changed. Therefore, changes of insulin and glucagon concentrations that regulate fibrinogen synthesis within a few hours (13, 14, 54) cannot be the reason for the lower rates of fibrinogen synthesis observed in our study. This was also in agreement with insulin data of a previous study with Intralipid in young male subjects (58). Absence of insulin elevation in our study indicated, in our opinion, that insulin resistance was not produced in our healthy subjects under the experimental conditions of moderate NEFA increase.
Furthermore, in dogs under pancreatic clamp condition, NEFA but not glycerol was shown to be linked to the regulation of hepatic glucose output (45, 46); therefore, regulation of the hepatic fibrinogen synthesis by parenteral administration of NEFA seems to be independent of the glucose homeostasis of the liver.
To our knowledge, there is no information about the possible mechanism that links the rapid increase of plasma NEFA levels to lower rates of fibrinogen synthesis. However, early energetic effects of NEFA do not explain the fast downregulation of fibrinogen synthesis, since the changes in the respiratory quotient that are indicative of preferential consumption of NEFA occurred several hours later.
A potential factor that might have influenced the results of our study seems to be glycerol. Glycerol has been infused as an osmotic, active additive of Lipofundin and was derived from the infused TG. The glycerol loading during the study was assessed to be 5.4 µmol·kg-1·min-1, which accounts for
2.7 µmol·kg-1·min-1 of the glucose formed by gluconeogenesis. In dogs, glycerol did not interfere with the insulin-controlled hepatic glucose output (46). Its potential effect on fibrinogen synthesis needs to be studied.
A further factor to be discussed is the degree of NEFA saturation. Saturated NEFA in vitro were found to stimulate fibrinogen synthesis more effectively than unsaturated and short-chain fatty acids, as shown by Pickart and Thaler (43), because unsaturated and short-chain NEFA are better substrates for oxidation (5, 6). However, the increase in fibrinogen synthesis is a late metabolic event and occurred in liver slices of mice (43) and in rabbits (42) 24 h after administration of TG. Therefore, oxidation of NEFA seems not to be involved in the early regulation of fibrinogen synthesis.
The object of our study has been the effects of NEFA on fibrinogen synthesis; metabolic pathways of other proteins were not evaluated. However, we consider inhibition of fibrinogen synthesis rate after NEFA to result from a specific regulatory process. Regulation of other proteins after NEFA elevation follows mechanisms distinctly different from that of fibrinogen. One example is the apolipoprotein B-100 synthesis for VLDL1 and VLDL2 secretion, which was increased after NEFA by 42.5 and 26.5%, respectively, in normal, glucose-tolerant individuals subjected to a protocol with Intralipid 30 that was similar to our study (44). The differences in regulation of the hepatic secretory proteins by NEFA may result from the intracellular compartmentation or interorgan zonation of the signal transduction (56).
Intra- and intercellular compartmentation of the liver is an accepted mechanism for regulating hepatic metabolism (28). With regard to carbohydrates, the hepatic cell metabolism around the afferent (periportal) vessels is different in capacity from cells located around the efferent (perivenous) vessels because of different exposure to signals by substrates, hormones, degradation products, oxygen gradient, or sympathetic nerve innervation (3, 29). Furthermore, the cross-talk between nonparenchymal and periportal and perivenous hepatocytes to control glucose supply, synthesis of reactive oxygen species, and gene transcription for synthesis of the type-2 acute-phase protein 2-macroglobulin (51) is an example of intercellular communication that is, however, not fully understood as yet (32). With regard to a possible link between NEFA elevation and fibrinogen synthesis, it should be considered that liver cells provide binding proteins for NEFA to control energy generation and host response (36), which may include synthesis of stress proteins such as fibrinogen. There is evidence that polyunsaturated fatty acids (n-3 family) upregulate the expression of genes that encode proteins that are involved in fatty acid oxidation and downregulate genes that encode proteins of lipid synthesis and sparing of glucose (8). This refers to polyunsaturated fatty acids as mediators of gene expression. It is not clear whether this concerns the DNA-protein interactions directly or whether it involves interactions with factors regulated by polyunsaturated fatty acids (9).
Lipid loading in fasted healthy volunteers of normal body weight results in moderate but significantly reduced fibrinogen synthesis, which in turn may cause lower plasma fibrinogen levels and reduced blood viscosity (30), i.e., a lipid-rich meal may not necessarily be associated with higher blood viscosity, since its elevation expected after lipid ingestion might be counter-acted by reduction of plasma fibrinogen synthesis. These acute effects of NEFA on fibrinogen synthesis, which is additive to the fibrinogen-lowering effect of insulin (13, 14) after a lipid-containing meal, may also occur after heavy, fat-rich meals.
Finally, our results do not explain the relation between chronically elevated plasma NEFA concentrations and equally increased fibrinogen concentration in insulin-resistant type 2 diabetes mellitus.
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LIMITATIONS OF THE STUDY |
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ACKNOWLEDGMENTS |
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This study was supported by Grant no. Fr 927/15 of the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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