Albumin, the most abundant protein in plasma, helps maintain colloid pressure of plasma and transports free fatty acids, bilirubin, and drug metabolites. The albumin gene is expressed in the liver and is under regulation at the transcriptional level. Tumor necrosis factor-α (TNF-α) mouse models have demonstrated that TNF-α acts through oxidative stress and nitric oxide pathways to inhibit albumin synthesis (1,2). Albumin production is decreased following exposure to TNF-α in human hepatoma cell lines (3). Although nutritional intake may affect albumin synthesis rates, infected rats had reduced albumin synthesis rates for 10 days following infection compared with pair-fed rats (4), suggesting that the inflammatory response had an effect on albumin synthesis beyond decreased nutritional intake. Children with active Crohn disease frequently have hypoalbuminemia, although the effect of anti-TNF-α therapy on albumin synthesis in these children is unknown.
Fibrinogen is a positive acute-phase protein that has an important role in thrombogenesis, and elevated plasma fibrinogen concentrations have been associated with cardiovascular disease (5). Crohn disease has been associated with an increased risk for thromboembolism (6). Fibrinogen levels have been found to be elevated in pediatric patients with Crohn disease, and tend to be higher in those with more active disease (7). Fibrinogen may induce the secretion of TNF-α by monocytes (8), but fibrinogen consumption may be increased in the presence of TNF-α (9). The direct administration of TNF-α to mice did result in increased serum fibrinogen, which was inhibited by polyclonal antibodies to murine TNF-α (10). The effect of anti-TNF-α therapy on fibrinogen synthesis in children with active Crohn disease is unknown.
Nutritional intake may alter albumin metabolism. The synthesis rate of albumin in normal subjects is increased by a liquid meal of glucose and lipids, and further increased with the addition of amino acids to the meal (11). Insulin may have a modulatory effect on albumin mRNA transcription (12). Similarly, short-term parenteral nutrition did not result in increased albumin synthesis rates in healthy humans (13). The provision of enteral nutrition did not result in changes in fibrinogen synthesis rates in normal subjects (11). However, in weight-losing cancer patients, fibrinogen synthetic rates increased in the enterally fed state, and the authors conclude that when an acute phase response is initiated by proinflammatory cytokines, the synthesis of acute phase reactants may be further increased by nutrition (14). The effect of parenteral nutrition supplementation on albumin and fibrinogen synthesis in children with active Crohn disease is unknown.
The aim of this study was to determine the effects of anti-TNF-α therapy on albumin and fibrinogen synthesis during both fasting and parenteral nutrition infusion in pediatric patients with active Crohn disease. We hypothesize that anti-TNF-α therapy will result in increased albumin synthesis and decreased fibrinogen synthesis. We also hypothesize that the provision of short-term parenteral nutrition will result in increased albumin and fibrinogen synthesis compared with the fasting state.
PATIENTS AND METHODS
We recruited children younger than 18 years of age with Crohn disease scheduled to receive their initial infusion of infliximab, an anti-TNF-α antibody. Patients were recruited from the outpatient Pediatric Gastrointestinal Disease Clinic of the James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis. These children were scheduled to receive an intravenous infusion of infliximab because of active Crohn disease unresponsive to conventional medical therapy, or complicated by fistula formation. Informed written consent was obtained from a legal guardian of each child, and each child provided written assent. This study was approved by the Indiana University–Purdue University Indianapolis and Clarian institutional review board, and by the Indiana University School of Medicine General Clinical Research Center advisory committee.
Stable Isotope Infusion
Children with active Crohn disease underwent outpatient metabolic assessment consisting of whole body protein metabolism immediately before and 2 weeks following initial infliximab infusion (15). Children were admitted to the General Clinical Research Center for metabolic assessment the morning after an overnight fast. An intravenous catheter was placed in a superficial vein for the purpose of drawing blood, and a baseline blood sample was obtained. A separate catheter was placed in the opposite extremity and a priming dose of stable isotope (in normal saline), representing 90 minutes of infusion, was administered for 5 minutes. Thereafter, constant infusion of [d5] phenylalanine (2.5 μmol · kg−1 · hour−1) dissolved in normal saline was delivered through the second venous catheter via an infusion pump. Blood samples (3 mL) were obtained at 120, 140, 160, and 180 minutes and frozen at −70°C for later analysis.
After the 180-minute sample was drawn, an intravenous solution of glucose (4.2 mg · kg−1 · minute−1), lipid (83 mg · kg−1 · hour−1) (Intralipid 20%, Baxter Healthcare, Deerfield, IL), and amino acids (63 mg · kg−1 · hour−1) (15% Clinisol, Baxter Healthcare) was infused for the remaining 2.5 hours of the study. If this infusion were to continue for 24 hours, it would have provided 48 kcal/kg, 6 g/kg of glucose, 2 g/kg of lipid, and 1.5 g/kg of protein. Blood samples (3 mL) were obtained at 270, 290, 310, and 330 minutes, and frozen for later analysis.
The Pediatric Crohn's Disease Activity Index (PCDAI) was administered to all patients at each of the 2 visits. The PCDAI includes subjective reporting of symptoms, physical examination findings including anthropometric measurements, and 3 laboratory examinations: hematocrit, erythrocyte sedimentation rate, and serum albumin. The PCDAI has been validated in children and adolescents (16).
Isolation of Albumin
Aliquots of 50 μL of plasma were mixed with 500 μL of 10% trichloroacetic acid. The samples were centrifuged at 13,000g for 5 minutes, and the supernatant was decanted. The pellet was resuspended with 500 μL absolute ethanol and sonicated for 30 minutes. The sample was recentrifuged at 13,000g for 5 minutes, and the supernatant was decanted and centrifuged at 13,000g until almost dry. The isolated albumin was resuspended in 200 μL nano-pure water. The solution was dried with a Speed-Vac Concentrator. The sample was hydrolyzed with 1 mL of 6 mol/L HCl for 18 hours at 110°C. The sample was dried in the Speed-Vac Concentrator.
Phenylalanine was decarboxylated to β-phenylethylamine by adding 1 mL of 0.5 mol/L trisodium citrate to dissolve the hydrolysate. Enzymatic decarboxylation was accomplished by adding 100 μL trisodium citrate buffer solution containing 0.25 U L-tyrosine decarboxylase and 0.25 mg pyridoxal phosphate. The solution was incubated at 50°C overnight, 100 μL of 6 mol/L NaOH was added, and the solution was centrifuged at 4000g for 10 minutes. The sample was extracted with 1 mL of diethyl ester. The diethyl ester layer was transferred to a new tube containing 200 μL of 0.1 mol/L HCl. The tube was shaken, and the ether phase was discarded. The sample was dried with the Speed Vac Concentrator. The sample was derivatized by adding 50 μL 50% N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide, and heated at 110°C for 1 hour.
Isolation of Fibrinogen
Aliquots of 200 μL of plasma were mixed with 100 μL of 0.5 mol/L CaCl and 2 mL of normal saline; 40 μL of thrombin (1 U/10 μL) was added to the tube and incubated at room temperature for 30 minutes. The tube was centrifuged at 1200g for 20 minutes. The supernatant was removed and discarded. The pellet was washed 3 times with 4 mL of normal saline and centrifuged at 1200g for 20 minutes each time. The pellet was hydrolyzed by adding 1 mL of 6 mol/L HCl and incubating at 110°C for 24 hours.
Following hydrolysis, 5 mL of water was added to the sample. A cation exchange column, using AG 50W X-8 resin (BioRad Laboratories, Hercules, CA), was prepared and washed with water. The samples were added to the column, and the column was washed with 8 mL of water. The phenylalanine was eluted with 2 mL of 6 mol/L NH4OH, and the sample was dried overnight in a Speed-Vac Concentrator. The sample was derivatized in an identical manner to the albumin samples.
Stable Isotope Analysis
The plasma enrichment of phenylalanine was determined by electron impact ionization and selected ion monitoring on a gas chromatograph-mass spectrometer (model 5973; Hewlett-Packard, Palo Alto, CA). The enrichment of plasma phenylalanine was determined by monitoring ions 178 and 183 after derivatization to the tertiary butyldimethylsilyl derivative. The final value for all determinations was corrected using an enrichment calibration curve.
Synthesis Rate Calculation
The slope of increasing albumin and fibrinogen enrichment with [d5] phenylalanine over time was calculated using the 4 plasma samples obtained during both the fasting and parenterally fed portions of the metabolic assessment. The slope was divided by the plasma enrichment of [d5] phenylalanine to calculate the fractional synthesis rate. The absolute synthesis rate was calculated by multiplying the fractional synthesis rate by the plasma concentration of albumin or fibrinogen and by 45 mL/kg, which estimates the plasma volume.
All of the results are reported as the mean ± standard error of measurement (SEM). Steady-state tracer enrichment was defined as an insignificant correlation (P > 0.05) with time. Comparisons within the sample group between pre- and postinfliximab measurements, and between fasting and parenterally fed measurements, were made using the paired t test. A P < 0.05 was considered statistically significant. A sample size of 15 was calculated to detect differences in whole body protein metabolism (15).
Serum Albumin and Fibrinogen
Fifteen patients completed the study, and complete information on subjects and clinical and biochemical outcomes previously have been published (15). The mean age of the subjects was 14.9 ± 0.3 years. Four of the 15 patients were receiving corticosteroids at the time of their initial infliximab infusion. The mean prednisone dose in these 4 patients was 33 mg/day at the time of the first infliximab infusion and 23 mg/day 2 weeks later at the time of the second infliximab infusion. The mean serum albumin changed from 3.56 ± 0.07 to 3.66 ± 0.04 (P = 0.10) 2 weeks following infliximab infusion (Fig. 1A). Fibrinogen data was available in 14 of 15 subjects. Mean serum fibrinogen decreased significantly from 230 ± 17 to 187 ± 8 mg/dL (P < 0.05) following a single dose of infliximab (Fig. 1B).
In the fasting state (Fig. 2A), there was no significant change in fractional albumin synthesis rate (percentage per day: preinfliximab = 8.2 ± 0.8, postinfliximab = 8.0 ± 0.9). Similarly, there was no change in the absolute albumin synthesis rate (milligrams per kilogram per day: preinfliximab = 132 ± 12, postinfliximab = 132 ± 15) (Fig. 2B). In the parenterally fed state (Fig. 2A), fractional albumin synthesis increased from 11.8% ± 1.3% to 15.1% ± 1.8%/day (P = 0.06) following a single dose of infliximab. The absolute albumin synthesis rate increased significantly from 191 ± 22 to 247 ± 28 mg · kg−1 · day−1 (P < 0.05) following infliximab (Figs. 2B and 3).
Because patients were evaluated during fasting and parenteral nutrition infusion at both the preinfliximab and postinfliximab time points, we were able to examine the effects of parenteral nutrition infusion on albumin synthesis rates. At both the preinfliximab and postinfliximab time points, the provision of parenteral nutrition resulted in a significant increase (P < 0.01) in albumin synthesis rates. At the preinfliximab time point, the addition of parenteral nutrition resulted in a 50% ± 15% increase in albumin synthesis. At the postinfliximab time point, the addition of parenteral nutrition resulted in a 107% ± 24% increase in albumin synthesis.
Despite a significant decrease in serum fibrinogen levels following infliximab therapy, we detected no significant changes in fasting or parenterally fed fractional and absolute fibrinogen synthesis rates following infliximab therapy. In the fasting state, the fractional fibrinogen synthesis rate changed from 25.9% ± 3.4%/day to 22.1 ± 2.2 %/day (Fig. 4A). The fasting absolute fibrinogen synthesis rate changed from 26.2 ± 3.9 mg · kg−1 · day−1 to 18.2 ± 1.7 mg · kg−1 · day−1 (Fig. 4B). During parenteral nutrition infusion, fractional fibrinogen synthesis rate changed from 26.0 ± 4.1 %/day to 30.5 ± 2.7 %/day (Fig. 4A). The absolute fibrinogen synthesis rate changed from 26.8 ± 4.7 mg · kg−1 · day−1 to 25.3 ± 2.3 mg · kg−1 · day−1 (Fig. 4B).
We compared fasting and parenterally fed fractional and absolute fibrinogen synthesis rates both before and after infliximab therapy. A nonsignificant mean increase in fibrinogen synthesis rate (46% ± 37%) was noted between fasting and parenterally fed states before infliximab therapy. Following the first dose of infliximab, however, the addition of parenteral nutrition resulted in a 77% ± 41% mean increase in fibrinogen synthesis rate (P < 0.05).
Contribution of Albumin and Fibrinogen to Total Protein Synthesis
We have previously published descriptions of changes in whole body protein synthesis following infliximab therapy in children with Crohn disease (15). Based upon the phenylalanine utilization rate for protein synthesis, total body protein synthesis in milligrams per kilogram per day can be calculated, and from this, the contributions of albumin and fibrinogen synthesis to this total amount can be assessed. In the fasting state, albumin synthesis accounted for 3.9% ± 0.5% and 5.0% ± 0.9% of total body protein synthesis before and after infliximab therapy, respectively, whereas fibrinogen synthesis accounted for 0.73% ± 0.09% and 0.62% ± 0.07% of total body protein synthesis, respectively. During parenteral nutrition infusion, albumin synthesis accounted for 4.1% ± 0.5% and 5.7% ± 0.7% of total body protein synthesis before and after infliximab therapy, respectively, while fibrinogen synthesis accounted for 0.62% ± 0.14% and 0.62% ± 0.09% of total body protein synthesis, respectively.
This is the first study of acute phase protein synthesis rates in children with Crohn disease, assessing the impact of both infliximab, an anti-TNF-α antibody, and parenteral nutrition. We have demonstrated increased albumin synthesis during the provision of intravenous nutrition following infliximab therapy in pediatric Crohn disease. Our study provides increased evidence that TNF-α inhibits albumin synthesis, especially in the anabolic state. In addition, although fibrinogen synthesis rates showed no significant change, a single dose of infliximab resulted in a significant decrease in serum fibrinogen levels. The fractional and absolute albumin and fibrinogen synthesis rates reported are similar to those found in previous studies in healthy young adults (17–19) and healthy children (20,21).
We previously have described changes in whole body protein metabolism in the same cohort of children (15). These children had significant decreases in proteolysis and protein synthesis following infliximab therapy, suggesting that active Crohn disease results in increased protein flux, as has been demonstrated in other studies (22). This reduction in protein synthesis may be due to decreased production of cytokines, inflammatory mediators, and positive acute phase proteins. A net reduction in protein synthesis does not preclude increased production of other specific proteins, such as albumin and other negative acute phase proteins, as we have demonstrated in this study. The contribution of albumin synthesis to whole body protein synthesis has been determined in healthy young adult subjects in both fasting (7%) and enterally fed (11%) states (23), and our results are similar, although slightly lower, which may be a result of our patients' age range or continued inflammation after the initial dose of infliximab therapy.
Compared with the fasting state, the provision of parenteral nutrition resulted in increases in both albumin and fibrinogen synthesis, reinforcing the effects of energy and protein intake on protein synthesis. The administration of nutritional support is given preferentially by enteral rather than parenteral nutrition. Enteral nutritional supplementation also may result in greater serum albumin levels than those found in patients receiving isocaloric, isonitrogenous parenteral nutrition supplementation, as has been reported in patients with active ulcerative colitis (24). Increased transcriptional regulators of albumin synthesis were found in rats receiving enteral nutrition compared with parenteral nutrition (25,26), suggesting the route of nutrition administration modulates regulation of albumin synthesis. Further studies of the effects of enteral nutrition supplementation on albumin and whole body protein synthesis in patients with inflammatory bowel disease are warranted.
We conclude that the administration of anti-TNF-α antibody and the provision of parenteral nutrition alter acute phase protein synthesis. By altering the effects of TNF-α, we have isolated changes in albumin, a critical nutritional protein. In the inflamed state, infliximab resulted in improved albumin synthesis during parenteral nutrition infusion, suggesting that the provision of nutrition and the attenuation of inflammation are important for maintaining normal albumin metabolism. This is of special importance in pediatrics, for which growth outcomes are critical in the treatment of inflammatory bowel disease. We also detected a significant change in serum fibrinogen levels following a single dose of infliximab, although the primary mechanism for this reduction does not appear to be a change in synthesis rate. Again, by altering the effects of TNF-α, there may be a decreased risk of thromboembolism and a reduction in long-term risk of cardiovascular disease. Similar changes in albumin and fibrinogen metabolism may be observed in other disease states with elevated TNF-α levels.
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