Introduction

The extreme hypermetabolic and hypercatabolic stress responses induced by a severe burn injury are characterized by increased proteolysis, lipolysis, and production of endogenous glucose via glycogenolysis and gluconeogenesis. The critical organ in the control of these processes is the liver. With major roles in metabolism, inflammation, immunity, and the acute phase response, the liver orchestrates the basic functions that modulate survival and recovery in severely burned patients. These authors, and others, have elucidated the function of the liver following a severe burn injury, and these studies have demonstrated that both the integrity of liver and the preservation of liver function are essential for survival. The expression of hepatic cytokines and acute phase proteins, which strongly correlates with post-burn survival, further supports this contention.

More than 2 million Americans each year experience a thermal injury – one of the most severe examples of a traumatic injury.¹ Worldwide, the World Health Organization (WHO) attributes >330 000 deaths each year to thermal injuries and their sequelae.² In the USA alone, more than 440 000 children are treated for burn injuries annually.³ Severe burn injuries – the third most common cause of death for American children – account for up to 1100 pediatric deaths each year,^4,⁵ accounting for a significant number of hospital admissions.^6,⁷ The effects induced by these injuries are not limited to the injured area alone. A severe burn injury has a devastating effect on the injured patient by affecting almost every organ system, resulting in heightened morbidity and mortality.⁸ Amplified availability of glucose leads to increased protein catabolism and lipolysis, initiating the post-burn hypermetabolic stress response.⁷^–⁹ Systemic inflammation, including pathophysiologic regulation of cytokines, hormones, and acute phase proteins, drives the hypermetabolic stress response.¹⁰^–¹² Prolongation or amplification of the hypermetabolic, inflammatory, or acute phase responses may result in dysregulation of counter-regulatory stress hormones (catecholamines, cortisol, glucagon), thereby exacerbating post-burn hypercatabolism, multi-organ failure, and death.¹¹^–¹³

For more than 20 years, focused research efforts on improving post-burn resuscitation, hypermetabolism, infection, ventilation, resuscitation, and wound healing have led to reduced morbidity and mortality.⁷ Further advances in clinical care, however, are needed to reduce morbidity and mortality even further. Through a series of studies, we have built a foundation that supports the hypothesis that the liver is a central player in the response to burn.¹⁴^–¹⁷ Through the modulation of immune, inflammatory, metabolic, and acute phase response signal transduction pathways, the liver contributes greatly to survival and recovery following a severe burn injury.¹³ Because of the dearth of information delineating the liver’s role after a thermal injury, we have spent the past 15 years engaging in studies to shed light on this topic. This chapter discusses liver function under normal conditions and following a severe insult such as a burn injury.

Anatomy, function, and physiology of the liver

Anatomy

In an average-sized adult, the liver weighs approximately 1500 g, making up almost 2% of total body weight. When the liver is injured, such as following a severe burn injury, its size can increase as needed to meet the additional demands. The lobar anatomy is used to divide the liver in the American system, using the intrahepatic location of the branches of the hepatic artery, portal vein and bile ducts. In this method, the left lobe is divided into medial and lateral sections, while the right lobe is divided into anterior and posterior sections. Soupault and Couinaud’s segmental system is used by the French to divide the liver into VIII segments. The hepatic artery and the portal vein are the two sources of the liver’s blood supply. One-quarter of the hepatic blood flow occurs via the hepatic artery, which provides oxygenated blood. Three-quarters of the blood flow through the liver is accounted for by the portal vein, which drains the splanchnic circulation.¹⁸

Physiology

A broad spectrum of biological functions is orchestrated by the liver. The fine interrelated physiologic-anatomic units of the liver direct these necessary processes: regulation of energy availability; nutrient management (including storage, distribution, and disposal); substrate synthesis, transformation, and metabolism; and metabolism and removal or toxins and pollutants.^18,¹⁹

1 The circulatory system. In order to build the pool of metabolites for energy and other functions, the liver extracts nourishment from the blood supply that extracts absorbed nutrients from the intestinal tract.

2 The biliary system. Bilirubin, cholesterol, detoxified drugs, and other liver-secreted substances are eliminated through the biliary system.

3 The mononuclear phagocyte system (MPS) (formerly known as the reticuloendothelial system, RES). A part of the immune system that detects antigens and disposes of senescent cells. Within the liver, Kupffer, endothelial, and phagocytic cells make up 60% of the MPS.

4 The hepatocytes. The metabolic demands of the entire body are met by the body. These cells synthesize, secrete, and store metabolites, and perform catabolic and anabolic functions. The generation of energy by converting adenosine triphosphate (ATP) to adenosine diphosphate (ADP) fuels local and systemic functions.

5 The liver as the source of hormones. The liver is the site of the synthesis and secretion of a variety of hormones including insulin-like growth factor-I (IGF-I), IGF binding proteins (IGFBPs), and hepatocyte growth factor (HGF). In addition to synthesizing and secreting necessary hormones, the liver acts as both a paracrine and an endocrine tissue, thereby amplifying the hepatic role in the hormonal axes.

Circulatory system

The majority of metabolic processes are regulated in one way or another by the liver. To fuel these functions, the liver expends 20% of the body’s total energy supply while consuming up to 25% of total utilized oxygen. These utilizations are not as high as one might expect due to an unfettered blood supply and the unique architecture within the liver. Estimations of the mean total hepatic blood flow range from 100 to 130 mL/kg per min. The majority (three-quarters) of the total hepatic blood flow is via the portal vein, while the remaining quarter is from the hepatic artery. When portal flow is reduced, a reciprocal increase in hepatic arterial blood flow follows; however, the reverse does not occur.^13,¹⁸ Plasma membrane-bound hepatocellular organelles enable specific functions while concurrently interacting with the extracellular matrix, facilitating exchange of metabolites between the blood compartment and the hepatocytes.^18,¹⁹ At the same time, the liver manufactures proteins, substrates, enzymes, and other substances for local use as well as for use by distal organs and tissues. Metabolic signals direct the liver to produce substrates needed to meet the energy requirements in other tissues. For example, the liver produces acetoacetate for utilization by brain, muscle, and the kidneys; however, this substrate is not used by the liver. Agonists, hormones, and substrates regulate the hepatic energy-related functions. As the liver receives blood via the arterial and portal circulation, nutrients are processed while toxins are metabolized, and then these are stored for later use, transformed for immediate use, or distributed to their final destination via lymphatic, vascular, or biliary circulations.

Portal venous flow into the liver is largely regulated by extrahepatic factors, including the flow rate from the intestines and spleen, food, bile salts, secretin, cholecystokinin, pentagastrin, epinephrine, vasoactive intestinal peptide, and glucagon. With blood making up 25–30% of the liver volume, the liver acts as a reservoir that can be tapped in time of physiologic need. More than 300 mL can be released into systemic circulation without adversely affecting liver function during acute blood loss. The liver can also store great amounts of blood – up to 1000 mL of blood can be taken up during right-sided heart failure without adversely affecting liver function.¹⁸^–²⁰

Biliary system

Although bile secretion occurs independently of liver blood flow, this is not the case when a patient is in shock. Bile is either formed at the hepatocyte’s canalicular membrane or by the bile ductules and ducts. Hepatocytes secrete the majority (four-fifths or approximately 1500 mL) of the total daily production of bile, while the bile duct epithelial cells secrete the remaining fifth. Phospholipids, proteins, conjugated bile acids, and cholesterol are the principal organic compounds in bile. Bile is modified by epithelial cells as it passes through the biliary ductules or ducts. Cholangiocytes, the highest cells lining the biliary ductules, seem to be hybrids of hepatocytes and ductular cells due to common functions and architecture. Bile secretion is stimulated by secretin, and the bile is secreted into the gallbladder where it is concentrated and stored under fasting conditions. Concentration of bile within the gallbladder is stimulated principally by cholecystokinin, with absorption of up to 90% of the water occurring within a 4-hour period. Bile is forced into the intestines following gallbladder contraction induced by cholinergic stimulation and subsequent relaxation of the sphincter of Oddi. The majority of the bile salt is absorbed into the enterohepatic circulation. Bile acids are then extracted by the liver and transported back to the canalicular membrane for re-secretion into the biliary system. Complete circulation of the total bile pool occurs 6–10 times/day in addition to 2–3 times per meal. Of the total 2–5 g pool, each day 0.2–0.6 g are lost in the stool; however, this is rapidly replaced by the synthesis of new bile acids.²⁰

When heme breaks down, bilirubin is produced and eliminated in the bile. Accumulation of bilirubin in the tissue or blood may occur with extrahepatic biliary obstruction or hepatocellular disease. The conjugation of bilirubin to albumin protects tissue from bilirubin’s toxicity until it is removed from the circulation by the liver via a carrier transport system. Bilirubin and glucuronide are conjugated within the hepatocyte, and then secreted into the bile. When this complex binds covalently with albumin, delta bilirubin is formed. Bacterial reduction of bilirubin in the intestine yields mesobilirubin and stercobilirubin, together known as urobilirubin, which are then excreted in the stool. Oxidation of urobilinogen to urobilin gives the stool its normal brown color.¹⁸^–²¹

Mononuclear phagocyte system (MPS)

The mononuclear phagocyte system (MPS), formerly known as the reticuloendothelial system (RES), is a component of the immune system and is made up of phagocytic cells, mainly monocytes and macrophages residing in the reticular connective tissue – such as the Kupffer cells in the liver. The Kupffer cells participate in the immune response by detecting foreign antigens and mobilizing the subsequent immune response. These cells further participate in immune function by removing and digesting senescent cells residing in the liver tissue.

Metabolic system

Acute phase response

In order to limit tissue damage and to initiate repair processes, the acute phase response (APR) is triggered following activating events such as a severe burn or trauma.^13,²² Pro-inflammatory cytokines are released locally by fibroblasts, endothelial cells, and activated phagocytic cells, which then leads to systemic dispersion of the APR.^13,²² The systemic phase of the APR induces a body-wide response encompassing the participation of many organs. The hypothalamus acts to raise body temperature, leading to fever. Steroid hormones are released by the pituitary–adrenal axis. Acute phase proteins are synthesized and secreted by the liver. Further promulgation of the hemopoietic responses is initiated in the bone marrow. Finally, the MPS involves the immune system in the APR, as the lymphocytes are activated to detect foreign antigens.^13,²² The interaction between the injury site and the liver, however, is a critical step in the initiation of the APR, as the liver is the primary site of acute phase protein production and modulation of the systemic inflammatory response. The APR is characterized by increased production of positive acute phase proteins such as C-reactive protein, α2-macroglobulin, and haptoglobin, with decreased expression of negative acute phase proteins, including albumin, transthyretin, transferrin, and retinol-binding protein.

Hormonal system

The liver is the site of hormonal synthesis, secretion, or interaction. Production and secretion into the bloodstream of angiotensinogen occurs within the liver. As noted above, Vitamin D is synthesized when cholecalciferol is hydroxylated by the enzyme 25-hydroxylase to become 25-hydroxycholecalciferol. Growth factors that are important for growth and development such as insulin-like growth factor-I (IGF-I) and the IGF binding proteins (IGFBPs) are made and secreted by the liver. Growth hormone (GH), the primary stimulus for the production of IGF-I, is produced by the pituitary gland but acts mainly on the hepatic cells (GH). Finally, synthesis of hepatocyte growth factor (HGF), a major hepatic regenerative growth factor, occurs in the liver.²⁷

The hepatic response to a severe thermal injury

Liver damage and morphological changes

Burn-induced liver injury is variable, and is typically proportional to the severity of the burn injury. Hepatomegaly, or a fatty liver, is a common post-burn finding (Fig. 25.1). These changes can be reversed; however, the significance of these alterations depends on the cause and the extent of the fat accumulation.²⁸ Autopsies of deceased pediatric burn victims have revealed that fatty infiltration of the liver is associated with increased bacterial translocation, liver failure, and endotoxemia.²⁹ How these fatty changes in the liver are induced by a burn injury is largely unknown; however, we posit that hepatic apoptosis (described in detail below) plays a major role in this development. Immediately following the thermal injury, increased hepatic edema is associated with damage to the liver. Jeschke et al.¹⁵ have shown that liver and body weight significantly increase at 2-7 days post-burn, as compared to non-burned liver/body weight. We also found that in burned rats, total hepatic protein concentrations were reduced significantly. These findings led to the notion that increased edema, not increased protein levels or hepatocyte numbers, underlies the increase in liver weight. This increase in edema, however, may induce release of hepatic enzymes into the circulation as a result of cellular damage or by altering membrane permeability. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are typically detected only at low levels in plasma. Therefore, their detection at elevated levels in the circulation are reliable indications of hepatocyte injury. Because elevations of AST can occur as a result of cardiac arrest or following muscle injury, ALT is considered to be a more specific test for hepatocyte injury. Severe hepatic damage can also be detected by elevations in serum glutamate dehydrogenase or alkaline phosphatase (ALKP). Elevations in ALKP provide insight into the function of the extrahepatic biliary tract as well and are frequently elevated in hepatobiliary disease. The liver damage induced by thermal injury is a result of the combination of edema formation, hypoperfusion, expression of pro-inflammatory cytokines and other cell death signals, and the release of the hepatic enzymes into the circulation. Following a severe burn, elevations between 50% and 200% above age-matched normals are recorded for serum AST, ALT, and ALKP (Fig. 25.2). These serum markers peak early – AST and ALT during the first post-burn day and ALKP during the second,^16,²⁶ indicating that burn-induced liver damage is a rapid phenomenon. During the acute hospitalization period, there is regeneration of the liver, which is apparent by the return of these enzymes to baseline. Although these enzymes are elevated as a direct result of the burn injury, additional physiologic processes may be amplifying their expression, including the elevation of ALKP occurring during growth spurts or alongside massive bone resorption. The careful evaluation of hepatic enzyme levels is warranted, and we recommend the comparison of multiple enzymes, as opposed to a single one, to determine whether liver damage is resolving.

Figure 25.1 (a) Massive hepatomegaly (upper) and hepatic fatty infiltration (lower) of a burn victim at autopsy. (b) Liver size increased throughout acute hospitalization by over 200% in 242 surviving burn patients.

(With permission from Jeschke MG. The hepatic response to thermal injury: is the liver important for postburn outcomes? Mol Med. 2009;15:337-351.)

Figure 25.2 Hepatic dysfunction of the rat burn model mimics the post-burn human disease state. (a) Serum aspartate amino transferase (AST) 24 and 48 h after thermal injury. (b) Serum alanine amino transferase (ALT) 24 and 48 h after thermal injury. (c) Serum albumin 24 and 48 h after thermal injury. (d) Caspase-3 activity in liver lysates as determined by successive Western blotting with active caspase-3 and actin antibodies. The data are expressed as a ratio of the two band intensities. (e) TUNEL staining of a liver section before (Control) and 24 h after (Burn) thermal injury. (f) Quantified TUNEL positive cells 24 and 48 h after thermal injury. Time after injury in hours is indicated. C, control; B, burn. Data presented are mean ± SEM. *p<0.05 (Burned animals n = 8 and controls n = 4 per time point). *Significant difference between burn and control, p<0.05.

(With permission from Jeschke MG. The hepatic response to thermal injury: is the liver important for postburn outcomes? Mol Med. 2009;15:337-351.)

Increases in hepatocyte death, both by apoptosis and necrosis, are associated with liver damage.¹⁵ Two distinctly different pathways result in cell death – programmed cell death (apoptosis) and necrosis.³⁰ Cell shrinkage, uniform fragmentation of DNA, and membrane blebbing characterize apoptotic cells, with the cell fragments being phagocytized by neighboring cells. Necrosis, on the other hand, is characterized by cellular swelling, fragmentation of the DNA in a random manner, activation of lysosomes, and complete breakdown of the cellular membrane enabling cellular contents to be extruded into the interstitium. These final steps induce an inflammatory response by attracting inflammatory cells, causing them to release free radicals and pro-inflammatory cytokines, leading to additional tissue breakdown. The morphological hallmarks unique to each process are used to differentiate between apoptotic and necrotic cells. At the time of autopsy, 10–15% of severely burned decedents had liver necrosis, as determined by pathological examination.²⁸ The hepatic necrosis related to burn-induced shock or sepsis was typically focal or zonal, central or paracentral, and sometimes microfocal.

Apoptosis also occurs in the liver following a cutaneous thermal injury (Fig. 25.2).¹⁵ The liver tries to maintain homeostasis when hepatocyte apoptosis increases by a compensatory increase in hepatocyte proliferation. Despite the attempt to maintain homeostasis in overall hepatocyte number, the liver is unable to immediately regain mass or maintain protein concentration. A severe thermal injury also can induce apoptosis of the epithelial cells in the small bowel.^31,³² Additionally, no increase is found in proliferation of the epithelial cells in the small bowel, resulting in a net loss of mucosal cells, and ultimately this is apparent as reduction in mucosal mass.^31,³² Cardiomyocytes are similarly affected by burn injury,^33,³⁴ although despite apoptosis of the cells, there is no compensatory increase in cardiocyte proliferation, leading to cardiac impairment and dysfunction.^33,³⁴

The molecular mechanisms that initiate and propagate hepatocyte apoptosis following a cutaneous burn are not known, although recent work associates hypoperfusion and ischemia-reperfusion with the induction of programmed cell death.³⁵^–³⁸ Blood flow to the bowel is decreased by approximately 60% for up to 4 hours following a thermal injury.³¹ Hepatic blood flow is likely decreased as well, and this may be one of the early events inducing programmed cell death. Apoptotic signals, including IL-1 and tumor necrosis factor (TNF)-α increase systemically during this same time frame.^30,³⁹^–⁴² Additional studies have revealed that these elevations of proinflammatory cytokines are not limited to the serum. Local elevations also occur after a thermal injury, as seen with increased concentrations of hepatic IL-1α, IL-1β, IL-6, and TNF-α.^14,⁴³^–⁴⁵ Taken together, these events – reduced splanchnic bloodflow and elevation of pro-inflammatory cytokines – are probably early events in the induction of hepatocyte apoptotic signaling.

Identifying molecular mechanisms

In identifying the molecular mechanisms that mediate the induction of hepatocyte apoptosis and dysfunction following a severe burn (Fig. 25.3), recent work has shown that ER stress is increased following a thermal injury and that cell-wide alterations in calcium are apparent with specific reductions in ER calcium that led to increases in cytosolic calcium.⁴⁶ Mitochondrial damage occurs as a result of increased cytosolic calcium, leading to the release of cytochrome c which then binds to the inositol triphosphate receptor (IP3R), inducing further depletion of calcium stores in the ER. ER stress and the unfolded protein response trigger apoptosis by activating JNK and initiating the downstream response. The serine on IRS-1 is then phosphorylated, blocking the tyrosine on the same protein from being activated by phosphorylation. At the same time, the pro-survival PI3K/Akt signaling pathway is blocked, amplifying the ER stress response by further activating the IP3R. If the unfolded protein burden can be limited through the use of chemical chaperones, this discovery may be of therapeutic significance as a method to promote hepatocyte survival.⁴⁷ Alternative pharmacologic agents are being developed to block pro-apoptotic ER stress signaling pathways, and, looking ahead, these alternatives may prove beneficial in the clinic by improving organ function and patient survival.⁴⁸