ORIGINAL ARTICLE: NUTRITION
Damaging Effects of Total Parenteral Nutrition Formula on Vascular Endothelium
Mehmet Demircan, Kubulay Gurunluoglu, Abdurrahman Karaman, and yBulent Mizrak
ABSTRACT Objectives: Sepsis in one of the most serious complications that can occur during total parenteral nutrition (TPN) procedures. In this experimental study, we investigated the effects of TPN, with or without lipid emulsion, on vascular endothelial damage. Methods: In total, 50 rabbits were used, divided into 5 groups of 10 each. TPN with lipids (group 1), TPN without lipids (group 2), and 0.09% saline (group 3) were given for 10 days via a central venous catheter. Group 4 received no treatment other than placement of a central venous catheter for 10 days. Group 5 was a control group. At the end of day 10, rabbits were sacrificed and tissue samples of liver, kidney, and inferior vena cava were prepared and examined by immunohistochemical methods for vascular cellular adhesion molecule (VCAM)-1 expression. Results: In tissue sections of liver, kidney, and inferior vena cava, VCAM-1 activity was increased prominently in TPN with and without lipids compared with the control group. VCAM-1 activity in the TPN with lipids group was decreased versus the TPN without lipids group (P > 0.05). Conclusions: The TPN procedure results in vascular endothelial cell damage not only in the vein where the solution is introduced but also in other parts of the vascular system. Even if it is not statistically significant, lipids in the TPN formula may decrease this endothelial cell damage, as shown by immunohistochemistry.
What Is Known
Endothelial damage occurs during total parenteral nutrition procedures at the vein where the solutions are introduced and damage is limited to that vein. The harmful effects of lipid emulsions to the immune system. Catheters have been blamed for causing endothelial damage that occurs with total parenteral nutrition procedures.
What Is New
Vascular endothelial damage does occur in total parenteral nutrition procedures, but it is not limited to the vein in which solutions are introduced, and may occur in veins throughout the body. Adding lipids to the total parenteral nutrition formulation may decrease this damage. The endothelial damage is ‘‘not’’ related to the catheter.
Key Words: lipid emulsions, sepsis, total parenteral nutrition, vascular endothelial damage, vascular cellular adhesion molecule-1
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T
otal parenteral nutrition (TPN) is a nutritional technique in which all nutritional requirements—such as water, proteins, carbohydrates, lipids, electrolytes, vitamins, and trace elements— are provided in appropriate preparations to the peripheral or central venous compartment, to maintain vital functions, and the anabolic milieu of the body, bypassing central regulatory mechanisms related to thirst and hunger (1). Parenteral nutrition was first described by Helfrik and Abelson at the beginning of the 1940s (2). Parenteral nutrition became much more important and widespread after 1969, when Dudrick
Received March 3, 2015; accepted April 14, 2015. From the ˙Ino¨nu¨ University Faculty of Medicine, Department of Pediatric Surgery, and yMedical Pathology, Malatya, Turkey. Address correspondence and reprint requests to Prof Dr Mehmet Demircan, MD, ˙Ino¨nu¨ University Medical School Department of Pediatric Surgery, Malatya 44315, Turkey (e-mail: [email protected]). This study was supported by TUBITAK (the Scientific and Technological Research Council of Turkey; Project number: 105S497, SBAG-HD-85). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000828
gave intravenous amino acids and hypertonic glucose solution to infants who could not be fed by the enteral route. But a substantial improvement was the discovery of lipid emulsions by Wretlind, because the patients did not survive on TPN without lipids (3,4). Indeed, with the widespread use of TPN in connection with pediatric gastrointestinal surgical operations, performed to correct congenital and acquired lesions, mortality decreased to a certain extent (5). TPN is, however, expensive, invasive, and nonphysiological; it also has many adverse effects. One of the most important complications that can occur during TPN practice is sepsis (6). Changes that occur in the vascular endothelium are of great importance for the development of sepsis (7). The endothelium, despite having a single layer and a seemingly ‘‘simple’’ structure, has many metabolic and regulatory functions between the blood and other tissues, has secretory functions, and is important in maintaining body homeostasis (8). The vascular endothelium mediates the relationship between the blood and other tissues (9). It provides control of vascular tone, cellular adhesion, inflammation, vascular permeability, and coagulation (10). To perform these tasks, cellular adhesion molecules are used (10). Adhesion molecules can be stored within the cell or can be synthesized when needed. Major adhesion molecules include the integrins, selectins, immunoglobulin supergene family members, and cadherins (10). Vascular cellular adhesion molecule-1 (VCAM-1), a member of the immunoglobulin supergene family, is an adhesion
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molecule that causes neutrophils to collect within an inflammatory area and to pass into tissues during sepsis (11,12). Many studies have examined how sepsis occurs during TPN practice (2,6,7). In this study we sought to explore the damaging effects of TPN formula on the vascular endothelium immunohistochemically and to assess the role of lipid emulsions in this phenomenon.
METHODS In this experimental study, 50 albino Angora rabbits, males and females, were used. Rabbits were obtained from the Inonu University Experimental Research Center. Approval was obtained from the Inonu University Medical Faculty Ethics Committee for Experimental Animals. The experimental setting was a room with a 12/12-h dark/light photoperiod at 238C. Rabbits were weighed before and after the experiment.
Method Experimental Groups The 50 rabbits were divided into 5 groups: 1. Group 1: 10 rabbits given TPN-containing lipids through a central venous catheter for 10 days. 2. Group 2: 10 rabbits given TPN without lipids through a central venous catheter for 10 days. 1 1 3. Group 3: 10 rabbits given 0.09% saline (50 mL kg day ) through a central venous catheter for 10 days. 4. Group 4: 10 rabbits given nothing through a central venous catheter for 10 days after the central catheterization procedure. 5. Group 5: 10 rabbits that underwent no treatment or catheterization. During the study, all rabbits continued to feed orally and were provided with drinking water. Each rabbit in groups 1 to 4 was catheterized with a 0.5 0.5-mm polyethylene catheter, 45 cm in length (Cavafix Certo; Braun, Melsungen, Germany), in the internal jugular vein, which was settled by a cutdown method. The procedure was performed under anesthesia (ketamine, 35 mg/kg IM, Ketalar; Parke-Davis, Ann Arbor, MI, and Xilazin, 5 mg/kg IM, Rompun; Bayer AG, Leverkusen, Germany).
TPN Formula The TPN formulation consisted of 10% lipid (Intralipid; Fresenius-Kabi, Uppsala Sweden), 6% amino acids (Trophamine; Eczacıbas¸ı-Baxter, Istanbul, Turkey), 20% dextrose (Dekstroz; Eczacıbas¸ı-Baxter), and trace elements (Addamel; Fresenius-Kabi). Characteristics of the daily TPN formulae with or without lipids that were introduced into the rabbits are shown in Table 1. All infusions started 1 day after the catheter was introduced and lasted 8 h/day for 10 days.
Tissue Samples At the end of the experimental period, rabbits were sacrificed and tissues samples were taken from the kidneys, the liver, and the inferior vena cava. The samples were divided into 2 pieces by transverse sectioning. Tissues materials were fixed in 10% formalin solution for 24 hours, then embedded in paraffin wax. Sections (5-mm thick) were prepared and evaluated by immunohistochemical methods for VCAM-1 expression. Sections were examined by 2 pathologists blinded to the groups. www.jpgn.org
Effects of TPN Formula on Vascular Endothelium TABLE 1. Features of daily TPN formulae with and without lipids
Ingredients 6% amino acids 20% dextrose 10 %lipid 0.9 %NaCl KCl Mg Trace elements Ca TPN with lipid TPN without lipid
Dose 4 g/kg 18 g/kg 2 g/kg 3 mEq/kg 3 mEq/kg 1 mEq/kg — 1 mEq/kg
Volume, mL
Calories, kcal/g
Osmolality, mOsm/L
160 250 50 50 3 1 1 1 516 466
40 170 55 — — — —
525 1250 280 310 — — —
265 210
851 913
TPN ¼ total parenteral nutrition.
Immunohistochemical Methods and Evaluation The inferior vena cava was evaluated as a representative large vein. Kidney and liver tissues were sampled to evaluate medium and small veins. To show VCAM-1 expression, sections on poly-Llysine–coated slides were used. Tissue sections were incubated for 1 hour at 608C. They were treated for 10 minutes with xylene and ethanols and washed for 1 to 2 minutes with distilled water. For antigen retrieval, sections were boiled in ethylenediaminetetraacetic acid solution, pH 8, for 20 minutes, then held at room temperature for 20 minutes, washed with phosphate-buffered saline (PBS), and then incubated with 3% hydrogen peroxide solution for 10 minutes. They were then washed in PBS and treated with blocker. The sections were then incubated with anti–VCAM-1 (mouse monoclonal antibody against rabbit VCAM-1; Labvision, Fremont, CA) for 60 minutes at room temperature. The sections were washed with PBS (3) and treated with biotinylated goat antibody for 20 minutes. The tissue sections were washed with PBS (3) and treated with streptavidin peroxidase for 20 minutes. The sections were washed with PBS (3), stained with the AEC chromogen for 20 minutes, counterstained with hematoxylin, and finally coverslipped using glycerin gel. The sections were evaluated by examination of 20 randomly chosen areas at 200 magnification. Immunohistochemical staining was classified as (0), no staining; (I), scant staining; (II), evident staining; and (III), diffuse staining.
Statistical Analysis Means and standard deviations of all data were calculated. Differences between the averages were evaluated by analysis of variance; differences between the groups were evaluated by least significant differences. All statistical analyses were performed using the SPSS software (SPSS Inc, Chicago, IL). P < 0.05 were considered to indicate statistical significance.
RESULTS The rabbits in all groups were weighed at the beginning (2600 500 g) and end (2550 475 g) of the experiment. The difference was not statistically significant (P > 0.05). Mean VCAM-1 activity levels in the endothelium of small veins of the liver and kidney and the inferior vena cava tissue sections are shown in Figure 1. Liver tissue sections in the 2 groups that received TPN with and without lipids showed prominently increased VCAM-1 activity in small veins versus the control group (1.5 0.2 vs 0.1 0.1 and 1.7 0.2 vs 0.1 0.1, respectively;
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2.5 2*
2
1.7*
VCAM
1.5*#
1.5
1.5*#
1.4*
1.3*#
1
0.7 0.7 0.7
0.5
0.3 0
0.1
0
0.2 0
0 Group 1
Group 2 VCAM liver
Group 3 VCAM kidney
Group 4
Group 5
VCAM IVC
*P < 0.05, in compaison with groups 3, 4, and 5. # P > 0.05, in compaison with group 2.
FIGURE 1. Mean vascular cellular adhesion molecule (VCAM)-1 activity levels in the endothelium of small veins of the liver and kidney, and the inferior vena cava (IVC) tissue sections.
FIGURE 3. Increased vascular cellular adhesion molecule-1 expression in the endothelial layer of small veins in kidney tissue sections in the total parenteral nutrition without lipids group (arrow).
P < 0.05). Moreover, VCAM-1 activities in the 2 TPN groups were higher than those in the catheter and saline groups (0.7 0.1 and 0.07 0.00, respectively; P < 0.05). Although the difference was not statistically significant, VCAM-1 activity in the small veins of liver tissue sections was lower in the TPN with lipid group versus the TPN without lipid group (P ¼ 0.052). Figure 2 shows VCAM-1 expression in the endothelial layer of small veins in liver tissue sections in the groups of TPN with lipids, TPN without lipid, and the controls. VCAM-1 activity in the small veins of kidney tissue sections was prominently increased in the TPN groups with and without lipids versus the control group (1.3 0.2 vs 0.2 0.1 and 1.4 0.2 vs 0.2 0.1, respectively; P < 0.05). Moreover, VCAM-1 activities obtained from these 2 TPN groups were higher than those of the catheter and saline groups (0.7 0.1 and 0.0 0.0, respectively; P < 0.05). VCAM-1 activity in small veins in kidney tissue sections was decreased in the TPN with lipids group versus the TPN without lipids group; however, the difference was not statistically significant (P ¼ 0.064). Figure 3 shows VCAM-1 expression in the endothelial layer of small veins in kidney tissue sections in the TPN with lipids, TPN without lipids, and saline groups.
Since 1969, when TPN was first used widely, mortality has decreased to a certain extent (5,13). Although this decrease made
FIGURE 2. Increased vascular cellular adhesion molecule-1 expression in the endothelial layer of small veins in liver tissue sections in the total parenteral nutrition with lipids group (arrow).
FIGURE 4. Increased vascular cellular adhesion molecule-1 expression in the endothelial layer of the inferior vena cava tissue sections in the total parenteral nutrition without lipids group (arrow).
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VCAM-1 activity in the inferior vena cava tissue sections was increased prominently in the TPN with and without lipid groups versus the control group (1.5 0.2 vs 0.0 0.0 and 2.0 0.2 vs 0.0 0.0, respectively). This increase was statistically significant (P < 0.05). Moreover, VCAM-1 activities in these 2 TPN groups were higher than those of the catheter and saline groups (0.7 0.1 and 0.3 0.1, respectively; P < 0.05). Although there was no VCAM-1 activity in the control group, the catheter and saline groups showed slight activity. In these sections, VCAM-1 activity in the TPN with lipids group was decreased versus that in the TPN without lipids group, but the difference was not statistically significant (P ¼ 0.056). Figure 4 shows VCAM-1 expression in the endothelial layer of the inferior vena cava tissue section in the TPN with lipids, TPN without lipids, and catheter groups.
DISCUSSION
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TPN more popular, complications occurring during TPN practice have emerged as an important problem. Hepatobiliary dysfunction has been emphasized as one of the most important complications and has been considered to be caused by the ingredients of TPN solutions. Indeed, this has been the focus of many investigations (14). Furthermore, sepsis, emerging at 0.5 to 2.0/1000 catheter-days in patients who have received TPN, is another important complication that increases mortality and morbidity rates (13). Catheters used for the procedure have been implicated in the pathogenesis of sepsis related to TPN practice (6). It was suggested that the catheters enabled microorganisms to enter and survive within the body (6). It has also been claimed that the lipids included in TPN formulations lead to sepsis by causing changes in the immune system (15). Both clinical and experimental studies have shown that the lipids within TPN formulae can cause immune response changes, depending on their chemical nature. Few studies of the overall effects of these changes and how they cause the development of sepsis have, however, been performed; the reported studies do not provide adequate data for any conclusion to be reached (16). Vascular endothelial damage occurring during TPN procedures is a factor in the sepsis development. Vascular endothelial damage has an important role in the development of sepsis (11). In researching vascular endothelial damage, it is important to measure adhesion molecule levels. VCAM-1, as used in our study, is one of the most frequently used adhesion molecules. Adhesion molecules become activated by mechanical and/or local trauma, resulting in endothelial damage (17). DeSouza et al (17) found that VCAM-1 was increased as a result of mechanical damage in which the endothelium had been exposed. They argued that this situation could be recovered with antihypertensive therapy. In another study, Guzman et al (18) applied trauma with balloon angioplasty to the aortas of rabbits and examined the tissues at 2 and 7 days following the trauma. They found an increase in VCAM-1 activity in the samples taken after 2 days and hyperplasia of the intimal layer, extracellular matrix deposition, and inflammatory cell infiltration after 7 days. De Caterina et al (19) argued that rabbits that were hypercholesterolemic first showed damage, then inflammation in their vascular endothelium, before developing atherosclerosis. They determined that the endothelial damage was caused by the increase in VCAM-1 activity. Likewise, Park et al (20) demonstrated that the accumulation of inflammatory cells in the early stages of atherosclerosis was the result of the induced expression of proinflammatory adhesion molecules, such as VCAM-1, and that this played an important role in the initiation of atherosclerosis. Several studies of the mechanism of vascular endothelial damage during TPN procedures have been reported (7,21,22). In 1 experimental study, Kuwahara et al (7) introduced into rabbits TPN solutions of different osmolarities and then performed a histopathological examination. They determined an increase in inflammatory cells and perivascular edema, and highlighted the pH and osmolarity of the solutions and the duration of the infusion as reasons (21). In a similar study, Terada et al (22) claimed that vascular endothelium damage emerging during TPN procedures was caused by the selenium and sulfhydryl groups present in TPN solutions. They cultured a human umbilical vein in tissue culture and treated it with selenium and sulfhydryl groups (as found in TPN); endothelial cell damage was evaluated by a histopathological examination. Saladino et al (23) examined the effects of lipids, used in TPN practice, on the arteries. They determined that 24 hours after the infusion, the vascular endothelium of the aortic arc and the thoracic aorta started to be damaged, because of the destruction of the subendothelial collagen; platelet adhesion had started and phagocytic cells were present around the intimal smooth muscle. In an experimental study, Nordstrand et al (24) showed that long-term www.jpgn.org
Effects of TPN Formula on Vascular Endothelium TPN practice resulted in inflammation in the pulmonary arteries, with no infection. In this study, we found that TPN formula caused damage to the endothelial cells of both large and small veins. The effects of parenteral nutrition are not limited to the vascular endothelium. In an experimental study by Dahl et al (25), it was determined that 1 day after TPN introduction, platelet aggregation was decreased, and after the third and seventh day, thrombocytopenia occurred. Porta et al (26) reported that the deterioration in platelet functions was not related to the lipids used in TPN. They used 2 different lipid emulsions and determined that there was no difference between them in terms of effects on platelet function. Although it has been claimed that the lipids in TPN have ‘‘negative’’ effects on the immune system, it has also been argued that they have ‘‘favorable’’ effects on the vascular endothelium. No previous study has, however, investigated the relationship between vascular endothelial damage and lipids using the VCAM-1 adhesion molecule. In our study, we showed that the damage in central and small veins caused by TPN formula could, at least in part, be prevented using lipids. Studies using different lipid emulsions examining the veins into which the lipids were introduced have reported similar results. In an experimental study, Bayer-Berger et al (27) introduced to rabbits 2 types of TPN formulation. The group that received 10% lipid had no vascular damage, whereas the other group received 20% lipid and showed endothelial damage. The authors reported that 10% lipid had a protective effect on the vessels. One important progress in TPN is the development of other lipid emulsions then Intralipid (or its equivalents). The oleic acid– rich newly developed emulsion and the Omegaven (with omega-3 fatty acids; Fresenius Kabi, Bad Homburg, Germany) may give totally different results than Intralipid based on soy oil. Serhan and his colleague Bannenberg have demonstrated that the importance of the balance between different fatty acids, that is, that omega-3 fatty acids can stop the inflammatory process, induced by omega-6 fatty acids (rich in soy oil and thus in Intralipid) by its derivates (28,29). They reviewed new cellular and molecular mechanisms for the resolution of inflammation, revealing key roles for eicosanoids such as lipoxins and recently discovered families of endogenous chemical mediators, termed resolvins and protectins. These mediators have anti-inflammatory and proresolution properties, thereby protecting organs from collateral damage, stimulating the clearance of inflammatory debris, and promoting mucosal antimicrobial defense (30). A few studies have examined the vascular endothelial damage caused by TPN procedures, not only in the vein where the TPN was introduced but also other systemic veins. Moreover, in 1 study, the aim was not to search for a relationship between vascular endothelial damage and sepsis, but the relationship with liver damage. In an experimental study, Tsuchioka et al (31) assessed the effects of TPN formula introduced into rats for 5 days. The small and middle veins in the liver were examined immunohistochemically for VCAM-1 expression, which was increased by endothelial damage. It was claimed that the endothelial damage caused inflammation in liver sinusoids, and that this inflammation resulted in damage to hepatocytes. They suggested that the liver damage occurring during the TPN procedure was related to the endothelial damage in the liver veins and that activation of VCAM1 disrupted immunomodulation by Kupffer cells, thereby causing inflammation in the sinusoids (31). Our experimental study showed that TPN formula resulted in damage to the endothelium of small, medium, and large veins. We consider that this damage may play a key role in the development of sepsis. Also we found that the lipids in the TPN formulation decreased the endothelial damage partially. Therefore, even if lipid emulsions have negative effects on immune responses, because they have positive effects on the vascular
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endothelium, they may be protective overall against the development of sepsis during TPN procedures. Many substances have toxic effects on vascular endothelial cells, including nickel ions found in orthodontic prostheses and vascular stents, beryllium used widely in industry, iron ions, and arsenic (15,32–34). Several substances in TPN solutions are toxic to these cells (22), including aluminum that has hepatotoxic effects, and selenium and sulfhydryl groups that have toxic effects on endothelial cells (1,22). Our results indicated that TPN formula may also contain other as-yet-unknown vascular toxic substances. There is also an issue as to whether endothelial cell damage can result in the development of sepsis versus depend on sepsis that already exists. This issue needs to be clarified because this is an important problem in patients with sepsis—should TPN be initiated or not? For that reason in our next study, we plan to assess TPN procedures with different types of lipid emulsion in a sepsis model that involves endothelial damage, and whether TPN affects the progression of sepsis and endothelial damage, in a group of subjects with endotoxemic sepsis. In conclusion, TPN procedures in rabbits resulted in vascular endothelial cell damage not only in the vein where the solution was introduced but also elsewhere in the vascular system. Although not statistically significant (borderline p-value), lipids in the TPN formulation may decrease this endothelial cell damage, as shown by immunohistochemistry. Vascular endothelial cell damage that occurs during TPN has no relationship with the catheter through which the solutions are administered. Instead, the damage results from the ingredients of the TPN formulation. Vascular endothelial cell damage may play a key role in the development of sepsis during TPN procedures. We consider that lipid emulsions may have protective effects against the development of sepsis during TPN procedures, because they have positive effects on the vascular endothelium.
REFERENCES 1. Demircan M, Ergun O, Coker C, et al. Aluminum in total parenteral nutrition solutions produces portal inflammation in rats. J Pediatr Gastroenterol Nutr 1998;26:274–8. 2. Teitelbaum DH, Coran AG. Nutrition. In: O’Neill JA, Rowe MI, Grosfeld JL, eds. Pediatric Surgery, 5th ed. New York: Elsevier: 171-96. 3. Wretlind A. Development of fat emulsions. J Parenter Enteral Nutr 1981;5:230–5. 4. Hallberg D, Holm I, Obel AL, et al. Fat emulsions for complete intravenous nutrition. Postgrad Med J 1967;43:307–16. 5. Vinnars E. Wilmore history of parenteral nutrition. J Parenter Enteral Nutr 2003;27:225–31. 6. Cercenado E, Ena J, Rodriguez-Creixems M, et al. A conservative procedure for the diagnosis of catheter-related infections. Arch Intern Med 1990;150:1417–20. 7. Kuwahara T, Asanami S, Kuba S. Experimental infusion phlebitis: tolerance osmolality of peripheral venous endothelial cells. Nutrition 1998;14:496–501. 8. Sader MA, Celermajer DS. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 2002;53:597–604. 9. Cantin M, Gutkowska J, Thibault G, et al. Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands. Histochemistry 1984;80:113–8. 10. Fishman AP. Endothelium: a distributed organ of diverse capabilities. Ann NY Acad Sci 1982;401:1–8. 11. Foster CA. VCAM-1/alpha-4-integrin adhesion pathway: therapeutic target for allergic inflammatory disorders. J Allergy Clin Immunol 1996;98:270–7.
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12. Chen CX, Yang LC, Xu XD, et al. Effect of propane-2-sulfonic acid octadec-9-enyl-amide on the expression of adhesion molecules in human umblical vein endothelial cells. Eur J Pharmacol 2015;756:15–21. 13. King DR, Komer M, Hoffman J, et al. Broviac catheter sepsis: the natural history of an iatrogenic infection. J Pediatr Surg 1985;20:728– 33. 14. Demircan M, Uguralp S, Mutus M, et al. The effects of acetylsalicylic acid, interferon-alpha, and vitamin E on prevention of parenteral nutrition-associated cholestasis: an experimental study. J Pediatr Gastroenterol Nutr 1999;28:291–5. 15. Seidner DL, Mascioli EA, Istfan NW, et al. Effects of long-chain triglyceride emulsions on reticuloendothelial system function in humans. J Parenter Enteral Nutr 1989;13:614–9. 16. Wanten G. An update on parenteral lipids and immune function: only smoke, or is there any fire. Curr Opin Clin Nutr Metab Care 2006;9:79– 83. 17. DeSouza CA, Dengel DR, Macko RF, et al. Elevated levels of circulating cell adhesion molecules in complicated essential hypertension. Am J Hypertens 1997;10:1335–41. 18. Guzman L, Mick M, Arnold A, et al. Role of intimal hyperplasia and arterial remodeling after balloon angioplasty: an experimental study in the atherosclerotic rabbit model. Arterioscler Thromb Vasc Biol 1996;16:479–87. 19. De Caterina R, Basta G, Lazzerini G, et al. Soluble vascular cell adhesion molecule-1 as a biohumoral correlate of atherosclerosis. Arterioscler Thromb Vasc Biol 1997;17:2646–54. 20. Park B, Yim J, Lee H, et al. Ramalin inhibits VCAM-1 expression and adhesion of monocyte to vascular smooth muscle cells through MAPK and PADI4-dependent NF-kB and AP-1 pathways. Biosci Biotechnol Biochem 2014;15:1–14. 21. Kuwahara T, Asanami S, Tamura T, et al. Dilution is effective in reducing infusion phlebitis in peripheral parenteral nutrition: an experimental study in rabbits. Nutrition 1998;14:186–90. 22. Terada A, Yoshida M, Seko Y, et al. Active oxygen species generation and cellular damage by additives of parenteral preparations: selenium and sulphydrile compounds. Nutrition 1999;15:651–5. 23. Saladino CF, Klein RA, Jonas EA. Induction of early atherosclerosis in rats using parentally-administered lipid emulsions. Artery 1987;14: 304–15. 24. Nordstrand K, Eide TJ, Almdahl SM, et al. Organ changes in pigs after long-term parenteral nutrition. Clin Nutr 1989;8:29–33. 25. Dahl PE, Osterud B, Kjaeve JC. Haematological disorders and lung alveolar macrophage function following total parenteral nutrition in rats. Clin Nutr 1992;11:269–76. 26. Porta I, Planas M, Padro JB, et al. Effect of two lipid emulsions on platelet function. Infusionsther Transfusionsmed 1994;21:316–21. 27. Bayer-Berger M, Chiolero R, Freeman J, et al. Incidence of phlebitis in peripheral parenteral nutrition: effect of the different nutrition solutions. Clin Nutr 1989;8:181–6. 28. Serhan CN. Resolution phases of inflammation: novel endogenous antiinflammatory and proresolving lipid mediators and pathways. Annu Rev Immunol 2006;25:101–37. 29. Serhan CN, Bannenberg G. Specialized pro-resolving lipid mediators in the inflammatory response: an update. Biochim Biophys Acta 2010;1801: 1260–73. 30. Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nature Rev Immunol 2008;8:349–61. 31. Tsuchioka T, Fujiwara T, Sunagawa M. Effects of glutamic acid and taurine on total parenteral nutrition. J Pediatr Surg 2006;41:1566–72. 32. Yao D, Shi W, Gou Y, et al. Fatty acid mediated intracellular iron translocation: a synergistic mechanism of oxidative injury. Free Radic Biol Med 2005;39:1385–99. 33. Togna G, Togna AR, Russo P, et al. Toxicological effects of beryllium on platelets and vascular endothelium. Toxicol Appl Pharmacol 1997;144: 262–7. 34. Grobe VJ. Peripheral circulatory disorders and acrocyanosis in arsenic exposed Moselle wine-growers. Berufsdermatosen 1976;24:78–84.
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Damaging Effects of Total Parenteral Nutrition Formula on Vascular Endothelium
Mehmet Demircan, Kubulay Gurunluoglu, Abdurrahman Karaman, and yBulent Mizrak
ABSTRACT Objectives: Sepsis in one of the most serious complications that can occur during total parenteral nutrition (TPN) procedures. In this experimental study, we investigated the effects of TPN, with or without lipid emulsion, on vascular endothelial damage. Methods: In total, 50 rabbits were used, divided into 5 groups of 10 each. TPN with lipids (group 1), TPN without lipids (group 2), and 0.09% saline (group 3) were given for 10 days via a central venous catheter. Group 4 received no treatment other than placement of a central venous catheter for 10 days. Group 5 was a control group. At the end of day 10, rabbits were sacrificed and tissue samples of liver, kidney, and inferior vena cava were prepared and examined by immunohistochemical methods for vascular cellular adhesion molecule (VCAM)-1 expression. Results: In tissue sections of liver, kidney, and inferior vena cava, VCAM-1 activity was increased prominently in TPN with and without lipids compared with the control group. VCAM-1 activity in the TPN with lipids group was decreased versus the TPN without lipids group (P > 0.05). Conclusions: The TPN procedure results in vascular endothelial cell damage not only in the vein where the solution is introduced but also in other parts of the vascular system. Even if it is not statistically significant, lipids in the TPN formula may decrease this endothelial cell damage, as shown by immunohistochemistry.
What Is Known
Endothelial damage occurs during total parenteral nutrition procedures at the vein where the solutions are introduced and damage is limited to that vein. The harmful effects of lipid emulsions to the immune system. Catheters have been blamed for causing endothelial damage that occurs with total parenteral nutrition procedures.
What Is New
Vascular endothelial damage does occur in total parenteral nutrition procedures, but it is not limited to the vein in which solutions are introduced, and may occur in veins throughout the body. Adding lipids to the total parenteral nutrition formulation may decrease this damage. The endothelial damage is ‘‘not’’ related to the catheter.
Key Words: lipid emulsions, sepsis, total parenteral nutrition, vascular endothelial damage, vascular cellular adhesion molecule-1
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T
otal parenteral nutrition (TPN) is a nutritional technique in which all nutritional requirements—such as water, proteins, carbohydrates, lipids, electrolytes, vitamins, and trace elements— are provided in appropriate preparations to the peripheral or central venous compartment, to maintain vital functions, and the anabolic milieu of the body, bypassing central regulatory mechanisms related to thirst and hunger (1). Parenteral nutrition was first described by Helfrik and Abelson at the beginning of the 1940s (2). Parenteral nutrition became much more important and widespread after 1969, when Dudrick
Received March 3, 2015; accepted April 14, 2015. From the ˙Ino¨nu¨ University Faculty of Medicine, Department of Pediatric Surgery, and yMedical Pathology, Malatya, Turkey. Address correspondence and reprint requests to Prof Dr Mehmet Demircan, MD, ˙Ino¨nu¨ University Medical School Department of Pediatric Surgery, Malatya 44315, Turkey (e-mail: [email protected]). This study was supported by TUBITAK (the Scientific and Technological Research Council of Turkey; Project number: 105S497, SBAG-HD-85). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000828
gave intravenous amino acids and hypertonic glucose solution to infants who could not be fed by the enteral route. But a substantial improvement was the discovery of lipid emulsions by Wretlind, because the patients did not survive on TPN without lipids (3,4). Indeed, with the widespread use of TPN in connection with pediatric gastrointestinal surgical operations, performed to correct congenital and acquired lesions, mortality decreased to a certain extent (5). TPN is, however, expensive, invasive, and nonphysiological; it also has many adverse effects. One of the most important complications that can occur during TPN practice is sepsis (6). Changes that occur in the vascular endothelium are of great importance for the development of sepsis (7). The endothelium, despite having a single layer and a seemingly ‘‘simple’’ structure, has many metabolic and regulatory functions between the blood and other tissues, has secretory functions, and is important in maintaining body homeostasis (8). The vascular endothelium mediates the relationship between the blood and other tissues (9). It provides control of vascular tone, cellular adhesion, inflammation, vascular permeability, and coagulation (10). To perform these tasks, cellular adhesion molecules are used (10). Adhesion molecules can be stored within the cell or can be synthesized when needed. Major adhesion molecules include the integrins, selectins, immunoglobulin supergene family members, and cadherins (10). Vascular cellular adhesion molecule-1 (VCAM-1), a member of the immunoglobulin supergene family, is an adhesion
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molecule that causes neutrophils to collect within an inflammatory area and to pass into tissues during sepsis (11,12). Many studies have examined how sepsis occurs during TPN practice (2,6,7). In this study we sought to explore the damaging effects of TPN formula on the vascular endothelium immunohistochemically and to assess the role of lipid emulsions in this phenomenon.
METHODS In this experimental study, 50 albino Angora rabbits, males and females, were used. Rabbits were obtained from the Inonu University Experimental Research Center. Approval was obtained from the Inonu University Medical Faculty Ethics Committee for Experimental Animals. The experimental setting was a room with a 12/12-h dark/light photoperiod at 238C. Rabbits were weighed before and after the experiment.
Method Experimental Groups The 50 rabbits were divided into 5 groups: 1. Group 1: 10 rabbits given TPN-containing lipids through a central venous catheter for 10 days. 2. Group 2: 10 rabbits given TPN without lipids through a central venous catheter for 10 days. 1 1 3. Group 3: 10 rabbits given 0.09% saline (50 mL kg day ) through a central venous catheter for 10 days. 4. Group 4: 10 rabbits given nothing through a central venous catheter for 10 days after the central catheterization procedure. 5. Group 5: 10 rabbits that underwent no treatment or catheterization. During the study, all rabbits continued to feed orally and were provided with drinking water. Each rabbit in groups 1 to 4 was catheterized with a 0.5 0.5-mm polyethylene catheter, 45 cm in length (Cavafix Certo; Braun, Melsungen, Germany), in the internal jugular vein, which was settled by a cutdown method. The procedure was performed under anesthesia (ketamine, 35 mg/kg IM, Ketalar; Parke-Davis, Ann Arbor, MI, and Xilazin, 5 mg/kg IM, Rompun; Bayer AG, Leverkusen, Germany).
TPN Formula The TPN formulation consisted of 10% lipid (Intralipid; Fresenius-Kabi, Uppsala Sweden), 6% amino acids (Trophamine; Eczacıbas¸ı-Baxter, Istanbul, Turkey), 20% dextrose (Dekstroz; Eczacıbas¸ı-Baxter), and trace elements (Addamel; Fresenius-Kabi). Characteristics of the daily TPN formulae with or without lipids that were introduced into the rabbits are shown in Table 1. All infusions started 1 day after the catheter was introduced and lasted 8 h/day for 10 days.
Tissue Samples At the end of the experimental period, rabbits were sacrificed and tissues samples were taken from the kidneys, the liver, and the inferior vena cava. The samples were divided into 2 pieces by transverse sectioning. Tissues materials were fixed in 10% formalin solution for 24 hours, then embedded in paraffin wax. Sections (5-mm thick) were prepared and evaluated by immunohistochemical methods for VCAM-1 expression. Sections were examined by 2 pathologists blinded to the groups. www.jpgn.org
Effects of TPN Formula on Vascular Endothelium TABLE 1. Features of daily TPN formulae with and without lipids
Ingredients 6% amino acids 20% dextrose 10 %lipid 0.9 %NaCl KCl Mg Trace elements Ca TPN with lipid TPN without lipid
Dose 4 g/kg 18 g/kg 2 g/kg 3 mEq/kg 3 mEq/kg 1 mEq/kg — 1 mEq/kg
Volume, mL
Calories, kcal/g
Osmolality, mOsm/L
160 250 50 50 3 1 1 1 516 466
40 170 55 — — — —
525 1250 280 310 — — —
265 210
851 913
TPN ¼ total parenteral nutrition.
Immunohistochemical Methods and Evaluation The inferior vena cava was evaluated as a representative large vein. Kidney and liver tissues were sampled to evaluate medium and small veins. To show VCAM-1 expression, sections on poly-Llysine–coated slides were used. Tissue sections were incubated for 1 hour at 608C. They were treated for 10 minutes with xylene and ethanols and washed for 1 to 2 minutes with distilled water. For antigen retrieval, sections were boiled in ethylenediaminetetraacetic acid solution, pH 8, for 20 minutes, then held at room temperature for 20 minutes, washed with phosphate-buffered saline (PBS), and then incubated with 3% hydrogen peroxide solution for 10 minutes. They were then washed in PBS and treated with blocker. The sections were then incubated with anti–VCAM-1 (mouse monoclonal antibody against rabbit VCAM-1; Labvision, Fremont, CA) for 60 minutes at room temperature. The sections were washed with PBS (3) and treated with biotinylated goat antibody for 20 minutes. The tissue sections were washed with PBS (3) and treated with streptavidin peroxidase for 20 minutes. The sections were washed with PBS (3), stained with the AEC chromogen for 20 minutes, counterstained with hematoxylin, and finally coverslipped using glycerin gel. The sections were evaluated by examination of 20 randomly chosen areas at 200 magnification. Immunohistochemical staining was classified as (0), no staining; (I), scant staining; (II), evident staining; and (III), diffuse staining.
Statistical Analysis Means and standard deviations of all data were calculated. Differences between the averages were evaluated by analysis of variance; differences between the groups were evaluated by least significant differences. All statistical analyses were performed using the SPSS software (SPSS Inc, Chicago, IL). P < 0.05 were considered to indicate statistical significance.
RESULTS The rabbits in all groups were weighed at the beginning (2600 500 g) and end (2550 475 g) of the experiment. The difference was not statistically significant (P > 0.05). Mean VCAM-1 activity levels in the endothelium of small veins of the liver and kidney and the inferior vena cava tissue sections are shown in Figure 1. Liver tissue sections in the 2 groups that received TPN with and without lipids showed prominently increased VCAM-1 activity in small veins versus the control group (1.5 0.2 vs 0.1 0.1 and 1.7 0.2 vs 0.1 0.1, respectively;
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2.5 2*
2
1.7*
VCAM
1.5*#
1.5
1.5*#
1.4*
1.3*#
1
0.7 0.7 0.7
0.5
0.3 0
0.1
0
0.2 0
0 Group 1
Group 2 VCAM liver
Group 3 VCAM kidney
Group 4
Group 5
VCAM IVC
*P < 0.05, in compaison with groups 3, 4, and 5. # P > 0.05, in compaison with group 2.
FIGURE 1. Mean vascular cellular adhesion molecule (VCAM)-1 activity levels in the endothelium of small veins of the liver and kidney, and the inferior vena cava (IVC) tissue sections.
FIGURE 3. Increased vascular cellular adhesion molecule-1 expression in the endothelial layer of small veins in kidney tissue sections in the total parenteral nutrition without lipids group (arrow).
P < 0.05). Moreover, VCAM-1 activities in the 2 TPN groups were higher than those in the catheter and saline groups (0.7 0.1 and 0.07 0.00, respectively; P < 0.05). Although the difference was not statistically significant, VCAM-1 activity in the small veins of liver tissue sections was lower in the TPN with lipid group versus the TPN without lipid group (P ¼ 0.052). Figure 2 shows VCAM-1 expression in the endothelial layer of small veins in liver tissue sections in the groups of TPN with lipids, TPN without lipid, and the controls. VCAM-1 activity in the small veins of kidney tissue sections was prominently increased in the TPN groups with and without lipids versus the control group (1.3 0.2 vs 0.2 0.1 and 1.4 0.2 vs 0.2 0.1, respectively; P < 0.05). Moreover, VCAM-1 activities obtained from these 2 TPN groups were higher than those of the catheter and saline groups (0.7 0.1 and 0.0 0.0, respectively; P < 0.05). VCAM-1 activity in small veins in kidney tissue sections was decreased in the TPN with lipids group versus the TPN without lipids group; however, the difference was not statistically significant (P ¼ 0.064). Figure 3 shows VCAM-1 expression in the endothelial layer of small veins in kidney tissue sections in the TPN with lipids, TPN without lipids, and saline groups.
Since 1969, when TPN was first used widely, mortality has decreased to a certain extent (5,13). Although this decrease made
FIGURE 2. Increased vascular cellular adhesion molecule-1 expression in the endothelial layer of small veins in liver tissue sections in the total parenteral nutrition with lipids group (arrow).
FIGURE 4. Increased vascular cellular adhesion molecule-1 expression in the endothelial layer of the inferior vena cava tissue sections in the total parenteral nutrition without lipids group (arrow).
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VCAM-1 activity in the inferior vena cava tissue sections was increased prominently in the TPN with and without lipid groups versus the control group (1.5 0.2 vs 0.0 0.0 and 2.0 0.2 vs 0.0 0.0, respectively). This increase was statistically significant (P < 0.05). Moreover, VCAM-1 activities in these 2 TPN groups were higher than those of the catheter and saline groups (0.7 0.1 and 0.3 0.1, respectively; P < 0.05). Although there was no VCAM-1 activity in the control group, the catheter and saline groups showed slight activity. In these sections, VCAM-1 activity in the TPN with lipids group was decreased versus that in the TPN without lipids group, but the difference was not statistically significant (P ¼ 0.056). Figure 4 shows VCAM-1 expression in the endothelial layer of the inferior vena cava tissue section in the TPN with lipids, TPN without lipids, and catheter groups.
DISCUSSION
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TPN more popular, complications occurring during TPN practice have emerged as an important problem. Hepatobiliary dysfunction has been emphasized as one of the most important complications and has been considered to be caused by the ingredients of TPN solutions. Indeed, this has been the focus of many investigations (14). Furthermore, sepsis, emerging at 0.5 to 2.0/1000 catheter-days in patients who have received TPN, is another important complication that increases mortality and morbidity rates (13). Catheters used for the procedure have been implicated in the pathogenesis of sepsis related to TPN practice (6). It was suggested that the catheters enabled microorganisms to enter and survive within the body (6). It has also been claimed that the lipids included in TPN formulations lead to sepsis by causing changes in the immune system (15). Both clinical and experimental studies have shown that the lipids within TPN formulae can cause immune response changes, depending on their chemical nature. Few studies of the overall effects of these changes and how they cause the development of sepsis have, however, been performed; the reported studies do not provide adequate data for any conclusion to be reached (16). Vascular endothelial damage occurring during TPN procedures is a factor in the sepsis development. Vascular endothelial damage has an important role in the development of sepsis (11). In researching vascular endothelial damage, it is important to measure adhesion molecule levels. VCAM-1, as used in our study, is one of the most frequently used adhesion molecules. Adhesion molecules become activated by mechanical and/or local trauma, resulting in endothelial damage (17). DeSouza et al (17) found that VCAM-1 was increased as a result of mechanical damage in which the endothelium had been exposed. They argued that this situation could be recovered with antihypertensive therapy. In another study, Guzman et al (18) applied trauma with balloon angioplasty to the aortas of rabbits and examined the tissues at 2 and 7 days following the trauma. They found an increase in VCAM-1 activity in the samples taken after 2 days and hyperplasia of the intimal layer, extracellular matrix deposition, and inflammatory cell infiltration after 7 days. De Caterina et al (19) argued that rabbits that were hypercholesterolemic first showed damage, then inflammation in their vascular endothelium, before developing atherosclerosis. They determined that the endothelial damage was caused by the increase in VCAM-1 activity. Likewise, Park et al (20) demonstrated that the accumulation of inflammatory cells in the early stages of atherosclerosis was the result of the induced expression of proinflammatory adhesion molecules, such as VCAM-1, and that this played an important role in the initiation of atherosclerosis. Several studies of the mechanism of vascular endothelial damage during TPN procedures have been reported (7,21,22). In 1 experimental study, Kuwahara et al (7) introduced into rabbits TPN solutions of different osmolarities and then performed a histopathological examination. They determined an increase in inflammatory cells and perivascular edema, and highlighted the pH and osmolarity of the solutions and the duration of the infusion as reasons (21). In a similar study, Terada et al (22) claimed that vascular endothelium damage emerging during TPN procedures was caused by the selenium and sulfhydryl groups present in TPN solutions. They cultured a human umbilical vein in tissue culture and treated it with selenium and sulfhydryl groups (as found in TPN); endothelial cell damage was evaluated by a histopathological examination. Saladino et al (23) examined the effects of lipids, used in TPN practice, on the arteries. They determined that 24 hours after the infusion, the vascular endothelium of the aortic arc and the thoracic aorta started to be damaged, because of the destruction of the subendothelial collagen; platelet adhesion had started and phagocytic cells were present around the intimal smooth muscle. In an experimental study, Nordstrand et al (24) showed that long-term www.jpgn.org
Effects of TPN Formula on Vascular Endothelium TPN practice resulted in inflammation in the pulmonary arteries, with no infection. In this study, we found that TPN formula caused damage to the endothelial cells of both large and small veins. The effects of parenteral nutrition are not limited to the vascular endothelium. In an experimental study by Dahl et al (25), it was determined that 1 day after TPN introduction, platelet aggregation was decreased, and after the third and seventh day, thrombocytopenia occurred. Porta et al (26) reported that the deterioration in platelet functions was not related to the lipids used in TPN. They used 2 different lipid emulsions and determined that there was no difference between them in terms of effects on platelet function. Although it has been claimed that the lipids in TPN have ‘‘negative’’ effects on the immune system, it has also been argued that they have ‘‘favorable’’ effects on the vascular endothelium. No previous study has, however, investigated the relationship between vascular endothelial damage and lipids using the VCAM-1 adhesion molecule. In our study, we showed that the damage in central and small veins caused by TPN formula could, at least in part, be prevented using lipids. Studies using different lipid emulsions examining the veins into which the lipids were introduced have reported similar results. In an experimental study, Bayer-Berger et al (27) introduced to rabbits 2 types of TPN formulation. The group that received 10% lipid had no vascular damage, whereas the other group received 20% lipid and showed endothelial damage. The authors reported that 10% lipid had a protective effect on the vessels. One important progress in TPN is the development of other lipid emulsions then Intralipid (or its equivalents). The oleic acid– rich newly developed emulsion and the Omegaven (with omega-3 fatty acids; Fresenius Kabi, Bad Homburg, Germany) may give totally different results than Intralipid based on soy oil. Serhan and his colleague Bannenberg have demonstrated that the importance of the balance between different fatty acids, that is, that omega-3 fatty acids can stop the inflammatory process, induced by omega-6 fatty acids (rich in soy oil and thus in Intralipid) by its derivates (28,29). They reviewed new cellular and molecular mechanisms for the resolution of inflammation, revealing key roles for eicosanoids such as lipoxins and recently discovered families of endogenous chemical mediators, termed resolvins and protectins. These mediators have anti-inflammatory and proresolution properties, thereby protecting organs from collateral damage, stimulating the clearance of inflammatory debris, and promoting mucosal antimicrobial defense (30). A few studies have examined the vascular endothelial damage caused by TPN procedures, not only in the vein where the TPN was introduced but also other systemic veins. Moreover, in 1 study, the aim was not to search for a relationship between vascular endothelial damage and sepsis, but the relationship with liver damage. In an experimental study, Tsuchioka et al (31) assessed the effects of TPN formula introduced into rats for 5 days. The small and middle veins in the liver were examined immunohistochemically for VCAM-1 expression, which was increased by endothelial damage. It was claimed that the endothelial damage caused inflammation in liver sinusoids, and that this inflammation resulted in damage to hepatocytes. They suggested that the liver damage occurring during the TPN procedure was related to the endothelial damage in the liver veins and that activation of VCAM1 disrupted immunomodulation by Kupffer cells, thereby causing inflammation in the sinusoids (31). Our experimental study showed that TPN formula resulted in damage to the endothelium of small, medium, and large veins. We consider that this damage may play a key role in the development of sepsis. Also we found that the lipids in the TPN formulation decreased the endothelial damage partially. Therefore, even if lipid emulsions have negative effects on immune responses, because they have positive effects on the vascular
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endothelium, they may be protective overall against the development of sepsis during TPN procedures. Many substances have toxic effects on vascular endothelial cells, including nickel ions found in orthodontic prostheses and vascular stents, beryllium used widely in industry, iron ions, and arsenic (15,32–34). Several substances in TPN solutions are toxic to these cells (22), including aluminum that has hepatotoxic effects, and selenium and sulfhydryl groups that have toxic effects on endothelial cells (1,22). Our results indicated that TPN formula may also contain other as-yet-unknown vascular toxic substances. There is also an issue as to whether endothelial cell damage can result in the development of sepsis versus depend on sepsis that already exists. This issue needs to be clarified because this is an important problem in patients with sepsis—should TPN be initiated or not? For that reason in our next study, we plan to assess TPN procedures with different types of lipid emulsion in a sepsis model that involves endothelial damage, and whether TPN affects the progression of sepsis and endothelial damage, in a group of subjects with endotoxemic sepsis. In conclusion, TPN procedures in rabbits resulted in vascular endothelial cell damage not only in the vein where the solution was introduced but also elsewhere in the vascular system. Although not statistically significant (borderline p-value), lipids in the TPN formulation may decrease this endothelial cell damage, as shown by immunohistochemistry. Vascular endothelial cell damage that occurs during TPN has no relationship with the catheter through which the solutions are administered. Instead, the damage results from the ingredients of the TPN formulation. Vascular endothelial cell damage may play a key role in the development of sepsis during TPN procedures. We consider that lipid emulsions may have protective effects against the development of sepsis during TPN procedures, because they have positive effects on the vascular endothelium.
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