There are very few studies regarding the levels of APP in PD and some of the results are apparently in conflict. Dufek and coworkers (2009) only found sporadic and slight abnormalities in the concentration of CRP and SAA in the serum of PD patients but McGeer and coworkers (2004), however, observed an increase of CRP in the affected dopaminergic areas of the brain. This apparent contradiction may be explained since the patients studied by Dufek and colleagues were moderately impaired and were all taking antiparkinsonian drugs, which may interfere in the peripheral APP measurements. In fact, a study of our laboratory shows that tHyc is increased in PD patients under L-dopa treatment (Martín-Fernández et al., 2010). On the other hand, other study regarding the APP levels in the blood of PD patients show an increase of CRP in the plasma according with the Hoehn-Yahr stages of PD severity (Seet et al., 2009). Importantly, in the study of Seet and coworkers the patients were advised to refrain from consuming their anti-parkinsonian medications 4 h before collection of the samples suggesting that a previous wash out of drugs may be needed in order to detect changes in these proteins. These results suggest that the dopaminergic loss in the brain is able to induce an increase of the peripheral APP that can be detected in the serum. In line with this, previous studies of our group have demonstrated that the specific degeneration of dopaminergic neurons induced by MPTP administration in non-human primate, stimulates an increase of CRP, SAA and Haptoglobin (HP) in plasma (de Pablos et al., 2009). This increase is very fast and transient, except for HP, which suggest that this response is related with the immediate effect of the toxin. On the other hand, a persistent increase of APP in the plasma suggests a persistent damage, which can be related with

The role of APP in PD is still unclear but increasing evidences show that the local inflammatory response in the brain is also reflected by several markers systemically and may be easily detected in the blood by well-established techniques. CRP seems to be a clear candidate as a PD biomarker but further studies are needed to soundly determine the specific factors that are increased in PD, which APPs are particularly increased and how are

Several inflammatory proteins, such as cytokines are linked with AD and some APP, such as tHcy and CRP, has been associated with the disease (Clarke et al., 1998). However, the relation between the APP and the presence of absence of dementia is still unclear. Very few studies report changes in the APP levels in VD and some of them present inconclusive results. Mancinella and coworkers (Mancinella et al., 2009) described that CRP levels are elevated in patients with VD respect to controls but still they show lower levels than patients with AD, which makes difficult to distinguish both disorders based in the peripheral biomarkers. Other authors did not find any differences in the CRP levels in VD but they however found an increase in tHcy (Davis et al., 2009). Importantly, high blood levels of tHcy have been related with cardiovascular diseases and may be also related with

they correlated with the dopaminergic degeneration and the level of Parkinsonism.

et al., 2009).

chronic degeneration.

**2.3 Vascular dementia and APP**

similar alterations in the VD (Lepara et al., 2009).

Table 1. Human Acute-Phase proteins.

Fig. 2. Different inflammatory triggering factors activate cells to stimulate local reactions. This response is mediated by different cytokines that are able to provoke a secondary systemic reaction characterized by the increase of leukocytes, activation of complement, increase of glucocorticoids in serum, reduction of Fe++ and Zn+ and the cascade of APPs.

Fig. 2. Different inflammatory triggering factors activate cells to stimulate local reactions. This response is mediated by different cytokines that are able to provoke a secondary systemic reaction characterized by the increase of leukocytes, activation of complement, increase of glucocorticoids in serum, reduction of Fe++ and Zn+ and the cascade of APPs.

Table 2. APP Classification production depending on the rate of their expression and normal concentration in human.

#### **3. Conclusions and therapeutic perspectives**

In neurodegenerative diseases changes observed blood parameters could be considered as putative hallmark of diseases evolution that might be helpful for personalized diagnosis, prognostic and/or progression diseases monitoring.

Obviously, the inflammatory response triggered in neurodegenerative diseases such as PD or AD is less easily detectable than those observed in other pathological conditions such as viral or bacterial infections. Accordingly, the APPs increase observed in neurodegenerative diseases is of less extent than the increase observed in infectious pathologies. Therefore, it is to be expected that the increase in APPs in patients exhibiting neurodegenerative diseases will be low as compared with other diseases. Although the relevance of APP measurements in the diagnosis of neurodegenerative diseases should need further evaluations, we believe that the prospects for blood-based biomarkers may be useful in the near future. We suggest that blood-based APP markers, perhaps in association with other biomarkers, will be useful tools for diagnosis of neurodegenerative diseases such as AD or PD and it is very likely that plasma markers will become part of the armamentarium for the investigation of CNS disorders.

The measurement of blood APP levels will be a safe and valuable tool for assessing and confirming the diagnosis of some disorders; the secretion of anti-inflammatory markers such as cytokines or APPs could be used to help to diagnose and evaluate the progression of the neurodegenerative process. However, more sensitive techniques of APP detection are needed, in particular to be used as putative diagnosis marker of neurodegenerative disorders.

#### **4. Acknowledgements**

This work was supported by grants from: the Spanish Ministry of Science & Innovation (SAF 2007-62262; FIS P10/02827), *Fundación Séneca* (FS/07/05662; FS/10/15329) and CIBERNED (*Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas*) to MTH. The authors would also like to thank Mr. P. Thomas for comments and language suggestions on this manuscript. Finally, the authors declare that there is no conflict of interest in the present work.

#### **5. References**

This work was supported by grants from: the Spanish Ministry of Science & Innovation (SAF 2007-62262; FIS P10/02827), *Fundación Séneca* (FS/07/05662; FS/10/15329) and CIBERNED (*Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas*) to MTH. The authors would also like to thank Mr. P. Thomas for comments and language suggestions on this manuscript. Finally, the authors declare that there is no conflict of

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### **The Role of the Acute-Phase Proteins in the Development and Progression of Liver Diseases**

Méndez Sánchez Nahum, Kobashi Margáin Ramón Arturo, Gavilanes Espinar Juan, Gutiérrez Grobe Ylse, Gutiérrez Jiménez Ángel and Uribe Misael *Liver Research Unit, Medica Sur Clinic & Foundation Mexico City Mexico*

#### **1. Introduction**

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The liver is a unique organ that is rich in cellular effectors for diverse systems, including the innate immune system, which plays a central role in hepatocellular injury (Gabay & Kushner, 1999). The innate immune system is based on broadly specific antigen recognition and does not rely on the more specialized antigen recognition pathways of the adaptive immune system. Although the innate immune response has broad specificity and is complex, acute-phase proteins (APPs) have been identified as biomarkers of the innate response (Table 1).

APPs are mostly synthesized in the liver. Their concentrations in plasma increase (positive APPs) or decrease (negative APPs) by at least 25% during inflammatory disorders. Most changes in APP concentration are due to infection, trauma, surgery, burns, tissue infarction, immunologically mediated or crystal-induced inflammation, and advanced cancer. Exercise, heatstroke, childbirth, or even psychological stress and some psychiatric disorders may also affect concentrations of these proteins. Cytokines are the chief stimulators and regulators of the production of these proteins. Interleukin (IL)-6 is the principal stimulator of APPs but other proinflammatory cytokines such as IL-1, tumor necrosis factor alpha (TNF-), interferon gamma (INF-), and transforming growth factor beta (TGF-), and chemokines such as IL-8 are also involved.

In the liver, the Janus kinase signal transducer and activator of transcription (STAT) pathway has been implicated in the action of cytokines. STAT 1 and STAT 2, which are activated by INF-/, are involved in antiviral defense and the former is involved in liver inflammation and injury. STAT 3 has been implicated in the acute-phase response as well as in hepatoprotective effects and liver regeneration. This pathway is mostly activated by IL-6. STAT 4 and STAT 6 are associated with the ischemia/reperfusion cycle as promoters of and protectors against injury, respectively, and STAT 5 plays an important role in the regulation of hepatic genes and growth factors. Several studies have confirmed that IL-6 and IL-22 (a related cytokine) are responsible for important liver functions such as liver regeneration, glucose and lipid metabolism, and induction of antiapoptotic proteins (Gao, 2005).

Table 1. Descriptive table of some of the positive and negative acute-phase proteins.

Many clinical applications are based on these proteins. The most widely used indicators of the APP response are erythrocyte sedimentation rate (ESR) and plasma C-reactive protein (CRP) concentration. The latter is considered more reliable because ESR changes slowly, whereas CRP concentration changes rapidly.

#### **2. Acute-phase proteins and the liver**

The synthesis of these proteins takes place, as mentioned before, in the liver, which is an organ with multiple implications for the metabolism of many compounds in the organism. It is logical to believe that any impairment in the functioning of this organ would affect the concentrations of and, therefore, the function of these proteins. In this chapter, the pathophysiology of these changes and the clinical repercussions thereof are discussed.

To begin this chapter, it is important to review information on one of the APPs that has been most associated with the liver. It is well known that albumin is the most abundant protein in the body and albumin concentration is often used to assess liver function as well as the nutritional state of the patient because a decrease in the level of this protein is often associated with liver dysfunction. As such, it is an important component in several scales of dysfunction and prognosis. Albumin level is a component of the Child–Turcotte–Pugh scale, the Model of End-stage Liver Disease scale (specifically designed for the liver), and global scales such as the Sepsis-related Organ Failure scale and the Acute Physiology and Chronic Health Evaluation scale. Some authors (Chan, 2010) have detected albumin mRNA in the blood, indicating that cell death could occur in liver impairment, which would release this element to the blood. Unlike albumin, the albumin mRNA level increases at an early stage of liver impairment. Research is needed to confirm this finding, but analysis of levels of circulating nucleic acids may be an interesting option for opportune detection of liver damage. Furthermore, in acute liver failure, the reduction in the production of albumin and 1-acid-glycoprotein alters the protein-bound fraction of many drugs, resulting in higher levels of the free and active forms of drugs and their associated effects. Nevertheless, albumin is not entirely specific for liver function impairment or injury; therefore, several other biomarkers have been investigated. These will be discussed in relation to the various liver pathologies.

#### **2.1 Alcoholic liver disease**

108 Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases

**ACUTE PHASE PROTEINS**

**Complement system** Albumin

**Coagulation and fibrinolytic system** Transthyretin

**Transport proteins** Insulin-like

**Ceruloplasmin, Haptoglobin, Hemopexin** Factor XII **Inflammation responders** Antithrombin

**Others α**1-Acid

Table 1. Descriptive table of some of the positive and negative acute-phase proteins.

Many clinical applications are based on these proteins. The most widely used indicators of the APP response are erythrocyte sedimentation rate (ESR) and plasma C-reactive protein (CRP) concentration. The latter is considered more reliable because ESR changes slowly,

The synthesis of these proteins takes place, as mentioned before, in the liver, which is an organ with multiple implications for the metabolism of many compounds in the organism. It is logical to believe that any impairment in the functioning of this organ would affect the concentrations of and, therefore, the function of these proteins. In this chapter, the pathophysiology of these changes and the clinical repercussions thereof are discussed. To begin this chapter, it is important to review information on one of the APPs that has been most associated with the liver. It is well known that albumin is the most abundant protein in the body and albumin concentration is often used to assess liver function as well as the nutritional state of the patient because a decrease in the level of this protein is often associated with liver dysfunction. As such, it is an important component in several scales of dysfunction and prognosis. Albumin level is a component of the Child–Turcotte–Pugh scale, the Model of End-stage Liver Disease scale (specifically designed for the liver), and global scales such as the Sepsis-related Organ Failure scale and the Acute Physiology and Chronic Health Evaluation scale. Some authors (Chan, 2010) have detected albumin mRNA in the blood, indicating that cell death could occur in liver impairment, which would release this element to the blood. Unlike albumin, the albumin mRNA level increases at an early stage of liver impairment.

**Antiproteases** Alpha-fetoprotein

**C3, C4, C9, Factor B, C1 INH, C4b-binding protein, Mannose binding**

**Fibrinogen, Plasminogen, Tissue plasminogen activator, Urokinase,**

**α1-Protease inhibitor, α1-Antichymotrypsin, Pancreatic secretory**

**Phospholipase A2, Lipopolysaccharide-binding protein, IL-1R**

**C-reactive protein, Serum amyloid A and P, Fibronectin, Ferritin,**

**Protein S, Vitronectin, Plasminogen activator inhibitor-1**

**trypsin inhibitor, Inter- α -trypsin inhibitors**

**antagonist, Granulocyte colony-stimulating factor**

**Angiotensinogen, α2-Macroglobulin**

whereas CRP concentration changes rapidly.

**2. Acute-phase proteins and the liver**

**lectin**

**POSITIVE NEGATIVE**

Transferrin

Thyroxin-binding

growth factor-1

Retinol-binding

glycoprotein

A2-HS glycoprotein

globulin

protein

Although liver disease may have many etiologies, alcohol is still the most frequent cause of liver deterioration, whether acute or chronic. The hepatotoxic threshold at which alcoholic liver disease (ALD) develops is 40 g of ethanol per day (about four drinks) for men and 20 g of ethanol per day (about two drinks) for women and it has been estimated that 7.4% of the adult population of the USA and 20–30% of the adult population of Europe consume high quantities of alcohol. It is alarming that, in these locations and in many developing countries, the increase in alcohol consumption has been continuous and is predicted to continue during the next decade. Figure 1 shows worldwide mortality due to alcoholic liver disease.

Fig. 1. Annual mortality from alcoholic liver disease. The countries with the highest mortality rates are shown.

Nevertheless, alcohol consumption is considered by many people to be normal and customary; this and the fact that patients usually do not seek medical assistance until the damage is well established (it is usually asymptomatic in the early stages) suggests that the prevalence of ALD is underestimated.

Several studies have proven that activation of Kupffer cells increases TNF- secretion, and therefore the immune response. Su and colleagues (Su et al., 1998) found in rat models that lipopolysaccharide, an endotoxin usually present in Gram-negative bacteria, is responsible for the activation of Kupffer cells. Lipopolysaccharide binding protein (LBP) opsonizes cells and presents the endotoxin to CD14, a membrane-bound glycoprotein that activates macrophages. Alcohol is one of the stimuli necessary to increase the level of LBP-1 and, therefore, mediates liver damage. Until recently, IL-17 was thought to play an important role in alcohol-induced liver damage by activating T-helper cells, which maintain and increase inflammatory damage.

Another APP that has been associated with alcohol is alpha-fetoprotein (AFP). Although AFP has been considered a biomarker for hepatocellular carcinoma (see later in this chapter), this can be misleading because AFP is also present in ALD.

#### **2.2 Liver steatosis**

Nonalcoholic fatty liver disease (NAFLD) is the most common metabolic cause of liver dysfunction worldwide. It is believed that 10–24% of the general population has NAFLD, most cases of which are asymptomatic. It is a form of chronic liver disease that encompasses a wide spectrum of conditions that range from deposits of lipids (liver steatosis) to liver damage caused by proinflammatory compounds (nonalcoholic steatohepatitis, NASH). The natural history of this pathology has been thoroughly studied and it has been reported that NAFLD represents the first step in a progression to NASH (10–20% of cases develop NASH), liver cirrhosis (3–5% of cases develop liver cirrhosis), and hepatocellular carcinoma (Figure 2).

Fig. 2. Natural history of metabolic liver disease. NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

Under normal conditions, the liver is often exposed to endo- and exotoxins, which initiate the innate immune response. TNF- target genes are normally expressed at minimal levels, preventing damage to the organ. Furthermore, it has been demonstrated that TNF- is responsible for promoting liver regeneration. NAFLD and NASH are characterized by an increase in free fatty acid levels and NASH is also accompanied by an increase in lipid peroxidation and high concentrations of TNF-, which cause mitochondrial dysfunction. Mitochondrial impairment renders the cell susceptible to TNF--induced cytotoxic damage.

Obesity has been considered a major risk factor for the development of NAFLD because it produces a proinflammatory state that gradually injures the liver. Nevertheless, authors such as Crespo and colleagues found that TNF- levels are significantly higher in patients with NASH than in obese patients without NAFLD, suggesting that the former patients have increased susceptibility to TNF--mediated injury, rather than there being a cause– effect relationship between the cytokine and NASH.

Although several biomarkers of hepatic fibrosis have been identified (as is discussed later in this chapter), there are no reliable markers for NAFLD or NASH. Alanine aminotransferase is considered a marker for liver injury, but obesity may reduce the level of alanine aminotransferase, rendering it misleading in patients with NASH. Another issue is that identification of alcoholic and nonalcoholic liver damage is difficult. Gamma-glutamyl transpeptidase (GGT) levels are commonly used for this purpose but, in cases of NASH, they have also led to uncertain conclusions. Another report (Ohtsuka, 2005) suggested that carbohydrate-deficient transferrin could be a marker for differentiating between NAFLD and NASH. Nevertheless, Yoneda and colleagues (Yoneda et al., 2008) found that, although CRP and amyloid A levels are often used to assess inflammatory injury to the liver in clinical practice, these APPs are members of the short pentrexin family, the levels of which are only elevated as a systemic response to inflammation, whereas the long pentrexin, PTX3, is rapidly induced in damaged tissue in a tissue-specific manner. Thus, they concluded that PTX3 could be a more important biomarker for NASH than for non-NASH cases of liver damage.

#### **2.3 Viral hepatitis**

110 Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases

Nevertheless, alcohol consumption is considered by many people to be normal and customary; this and the fact that patients usually do not seek medical assistance until the damage is well established (it is usually asymptomatic in the early stages) suggests that the

Several studies have proven that activation of Kupffer cells increases TNF- secretion, and therefore the immune response. Su and colleagues (Su et al., 1998) found in rat models that lipopolysaccharide, an endotoxin usually present in Gram-negative bacteria, is responsible for the activation of Kupffer cells. Lipopolysaccharide binding protein (LBP) opsonizes cells and presents the endotoxin to CD14, a membrane-bound glycoprotein that activates macrophages. Alcohol is one of the stimuli necessary to increase the level of LBP-1 and, therefore, mediates liver damage. Until recently, IL-17 was thought to play an important role in alcohol-induced liver damage by activating T-helper cells, which maintain and

Another APP that has been associated with alcohol is alpha-fetoprotein (AFP). Although AFP has been considered a biomarker for hepatocellular carcinoma (see later in this

Nonalcoholic fatty liver disease (NAFLD) is the most common metabolic cause of liver dysfunction worldwide. It is believed that 10–24% of the general population has NAFLD, most cases of which are asymptomatic. It is a form of chronic liver disease that encompasses a wide spectrum of conditions that range from deposits of lipids (liver steatosis) to liver damage caused by proinflammatory compounds (nonalcoholic steatohepatitis, NASH). The natural history of this pathology has been thoroughly studied and it has been reported that NAFLD represents the first step in a progression to NASH (10–20% of cases develop NASH), liver cirrhosis (3–5% of cases develop liver cirrhosis), and hepatocellular carcinoma

Fig. 2. Natural history of metabolic liver disease. NAFLD, nonalcoholic fatty liver disease;

chapter), this can be misleading because AFP is also present in ALD.

prevalence of ALD is underestimated.

increase inflammatory damage.

NASH, nonalcoholic steatohepatitis.

**2.2 Liver steatosis**

(Figure 2).

Infection with the hepatitis C virus (HCV) is a leading cause of chronic liver disease. According to the World Health Organization, over 3% of the world's population (about 180 million people) is infected and about 130 million are at risk of developing cirrhosis. The majority of those infected with HCV (60–80%) develop chronic hepatitis, which is associated with progressive fibrosis. Of chronic hepatitis patients, 3–9% develop cirrhosis within 20 years. El-Serag and colleagues (El-Serag et al., 2003) found that HCV-infected patients are more frequently coinfected with viral or bacterial entities than are patients with other liver diseases. Few studies have been performed to explain this finding, but Wegert and colleagues (Wegert, 2009) found that CRP, SAA, haptoglobin, and fibrinogen levels are diminished secondary to an impairment caused by an HCV core protein that inhibits activation of transcription factors for protein production and disrupts TNF- and IL-6 signaling, affecting the whole immune response, at least in mice. Complement activity has also been suggested as an inducer of fibrosis and, therefore, of progression to cirrhosis in HCV-infected patients. It was reported that C5 is associated with this progression (Hillebrandt, 2005), but subsequent studies have failed to demonstrate this relationship; thus, it is unclear if this component is relevant to the induction of fibrosis in humans (Halangk, 2008). Despite this, the involvement of complement in the progression of fibrosis is still a topic of research. Interestingly, HCV-infected patients also have significantly lower cholesterol levels than normal subjects. This may be associated with APP level, which, as mentioned before, is reduced, as in the proinflammatory and proatherogenic states.

On the other hand, even though vaccination has considerably diminished the number of cases of HBV infection, the prevalence of this disease is high. About 350 million people worldwide are infected and are at risk of progression to a worse state (Te, 2002). Some authors have found that the response to the vaccine is closely related to complement activity. C4, a crucial protein for the classical complement pathway, links innate immunity with adaptive immunity by targeting the antigen to follicular dendritic cells and B cells through the specific receptors, CD21 and CD35. It has been shown that patients who do not show the expected response to the vaccine have human leukocyte antigen class-II alleles, which impair C4 activity. Vaccines should be linked to complement fragments to avoid this problem.

Both HCV and HBV infections are considered chronic. This characteristic enables a proinflammatory state to exist, which will eventually affect the synthesis of APP in the liver. Although other hepatotropic infections may also produce an acute state, it is usually selflimited and therefore the levels of APP do not change markedly.

#### **2.4 Liver cirrhosis**

Fibrosis is a type of tissue repair that is characterized by the replacement of normal parenchyma with connective tissue (collagen) and is mainly secondary to chronic inflammation. Fibroblasts and myofibroblasts are partly responsible for this process, and it is important to note that the principal stimulus for these cells is IL-6, which is initially secreted by macrophages and subsequently secreted by B cells at the site of the lesion. When inflammation is chronic, the stimulus is persistent; thus, abundant amounts of collagen are synthesized, resulting in abnormal accumulation of connective tissue. The gold standard for the diagnosis of cirrhosis is still the liver biopsy. Several scoring systems have been developed to stage cirrhosis. The Metavir scoring scale is most commonly used to indicate the grade of inflammation and scarring on the liver and was first developed for HCVinfected patients. The Knodell score is a histological staging system that assesses the extent of inflammation: a score of 0–10 is allocated to periportal or bridging necrosis and a score of 0–4 is allocated to intralobular degeneration and portal inflammation. The various grades used in these two scoring systems are shown in tables 2 and 3.

Table 2. The METAVIR scoring system for cirrhosis.

Table 3. The Knodell scoring system for cirrhosis.

cholesterol levels than normal subjects. This may be associated with APP level, which, as

On the other hand, even though vaccination has considerably diminished the number of cases of HBV infection, the prevalence of this disease is high. About 350 million people worldwide are infected and are at risk of progression to a worse state (Te, 2002). Some authors have found that the response to the vaccine is closely related to complement activity. C4, a crucial protein for the classical complement pathway, links innate immunity with adaptive immunity by targeting the antigen to follicular dendritic cells and B cells through the specific receptors, CD21 and CD35. It has been shown that patients who do not show the expected response to the vaccine have human leukocyte antigen class-II alleles, which impair C4 activity. Vaccines

Both HCV and HBV infections are considered chronic. This characteristic enables a proinflammatory state to exist, which will eventually affect the synthesis of APP in the liver. Although other hepatotropic infections may also produce an acute state, it is usually self-

Fibrosis is a type of tissue repair that is characterized by the replacement of normal parenchyma with connective tissue (collagen) and is mainly secondary to chronic inflammation. Fibroblasts and myofibroblasts are partly responsible for this process, and it is important to note that the principal stimulus for these cells is IL-6, which is initially secreted by macrophages and subsequently secreted by B cells at the site of the lesion. When inflammation is chronic, the stimulus is persistent; thus, abundant amounts of collagen are synthesized, resulting in abnormal accumulation of connective tissue. The gold standard for the diagnosis of cirrhosis is still the liver biopsy. Several scoring systems have been developed to stage cirrhosis. The Metavir scoring scale is most commonly used to indicate the grade of inflammation and scarring on the liver and was first developed for HCVinfected patients. The Knodell score is a histological staging system that assesses the extent of inflammation: a score of 0–10 is allocated to periportal or bridging necrosis and a score of 0–4 is allocated to intralobular degeneration and portal inflammation. The various grades

**2** Scarring has occurred and extends to blood vessels

mentioned before, is reduced, as in the proinflammatory and proatherogenic states.

should be linked to complement fragments to avoid this problem.

limited and therefore the levels of APP do not change markedly.

used in these two scoring systems are shown in tables 2 and 3.

**Grade Description 0** No scarring **1** Minimal scarring

**3** Bridging fibrosis

Table 2. The METAVIR scoring system for cirrhosis.

**Score Description 0** No inflammation **1–4** Minimal inflammation **5–8** Mild inflammation **9–12** Moderate inflammation **13–18** Marked inflammation

Table 3. The Knodell scoring system for cirrhosis.

**4** Advanced scarring, cirrhosis

**2.4 Liver cirrhosis**

Although the gold standard for the diagnosis of cirrhosis is the liver biopsy, the invasive nature of this procedure makes it difficult to perform with every patient. Although noninvasive methods such as the FibroScan and FibroTest are available, they may result in false-negative results in obese patients or patients with ascites. Although CRP and serum amyloid A (SAA) levels are biomarkers for liver injury and inflammation, it has been demonstrated that they are not correlated with fibrosis. Fortunately, there are other biomarkers that have been validated in several studies. Alpha-2 macroglobulin (A2M) is a protein that inactivates proteinases and inhibits fibrinolysis by reducing plasmin and kallikrein expression; thus, it inhibits the catabolism of matrix proteins, facilitating the development of fibrosis. The FibroTest involves this biomarker (Franciscus, 2010). The level of apolipoprotein A1 (ApoA1), a structural component of high-density lipoprotein cholesterol, is also correlated with the advanced stages of fibrosis (F3–F4). Other authors (Ho et al., 2010) described a novel biomarker: vitamin D binding protein (VDBP). Although A2M expression is upregulated in fibrotic livers, both ApoA1 and VDBP are negative APPs and are downregulated in fibrotic livers.

As mentioned in the introduction, there are several signaling pathways that control diverse functions. In the case of fibrosis, the STAT 3 family has the greatest impact. Activation and high activity of this signaling pathway have been implicated in the genesis of cirrhosis as well as in its evolution to HCC. Under normal circumstances, insulin-like growth factor-1 (IGF-1) maintains the activity of the STAT 5 signaling pathway; nevertheless, when liver damage occurs, growth hormone is released, with consequent lowering of IGF-1 level (therefore, it is known as a negative APP), which stops the activation of the STAT 5 pathway and the phosphorylation of STAT 3. TGF- is a closely related cytokine because it is also secreted when liver damage occurs and it activates the STAT 3 pathway as well as the expansion of T-helper cells, which increases fibrosis.

It has also been reported (Parsian et al., 2010) that expression of other components such as hyaluronic acid, alpha glycosaminoglycan distributed in extracellular spaces and synthesized by hepatic stellate cells in the liver, and laminin, a basal membrane glycoprotein synthesized in hepatocytes, also increase in the early stages of fibrosis in chronic liver diseases. These serum parameters have been compared with APP-based indices such as A1 apolipoprotein level and prothrombin time, and it was found that hyaluronic acid is the most strongly correlated with liver fibrosis.

#### **2.5 Hepatocellular carcinoma and other liver tumors**

As mentioned in the discussion of cirrhosis, downregulation of the STAT 5 signaling pathway and activation of the STAT 3 pathway increase fibrosis. Practically all chronic liver diseases result in fibrosis and, if not controlled, in malignant transformation of hepatocytes, and HCC (figure 3).

HCC accounts for 5.6% of all human cancers, more so among men than women (7.5% and 3.5%, respectively), and its prevalence increases with age. It has a 5-year survival rate of 6.5% and is considered responsible for 660,000 deaths per year worldwide. Viral hepatitis, alcohol, oral contraceptives, and aflatoxins are the most important risk factors for its development and its prevalence is expected to increase in the future.

HCV and HBV infection are the main causes of HCC; they are responsible for 80% of HCC cases (96% in HBV endemic regions). It has been established that 10–40% of all chronic HBV patients will develop this entity. The progression to liver cancer is monitored using serum levels of alpha-fetoprotein (AFP), an oncofetal glycoprotein that serves as an APP for several

Fig. 3. Evolution and risk factors for the development of hepatocellular carcinoma.

liver diseases; nevertheless, it is not cancer specific. Comunale and colleagues (Comunale et al., 2010) identified more than 100 glycoproteins that are elevated in cirrhotic and HCC patients and that may serve as markers of the early development of HCC, and they suggested that alpha-1-antitrypsin was the most promising.

CRP and SAA levels are correlated with tumor activity (Jong, 2001), i.e., with increasing tumor load, necrotic tumors, and tissue destruction. Levels of these proteins are even increased by gastric and colorectal metastases. It has also been suggested that APPs provide an ideal environment for tumor recurrence or growth (Harimoto et al., 2009). It is also accepted that CRP-positivity in HCC patients often indicates a poor prognosis and portal vein invasion.

As mentioned earlier in the chapter, hypoalbuminemia was considered a marker of liver impairment. For a long time, it was thought that tumor activity was responsible for the decrease in albumin level; however, recent studies (Al-Shaiba et al., 2004) have demonstrated that the reduction in serum albumin level is related to the inflammatory response rather than to nutritional depletion caused by the tumor.

#### **2.6 Autoimmune hepatitis**

Autoimmunity is also an important cause of liver disease. It encompasses a broad spectrum of diseases, including autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and autoimmune cholangitis. The most common are AIH and PBC. Clinical, biochemical, histopathological, and cholangiographic criteria are used for differential diagnosis (Table 4).

AIH is characterized by severe liver damage with a modest or low elevation of alkaline phosphatase level (Oo, 2010). It is classed into three types according to the autoantibodies expressed: type 1, smooth muscle antibodies (SMA) or anti-nuclear antibodies (ANA) are present; type 2, anti-liver kidney microsomal antibodies (LKM) are present; and type 3, soluble liver antibodies (SLA) or liver/pancreas antigens are present. All of these antibodies induce proinflammatory cells that mostly activate T-helper cells, which cause liver damage.

Fig. 3. Evolution and risk factors for the development of hepatocellular carcinoma.

suggested that alpha-1-antitrypsin was the most promising.

response rather than to nutritional depletion caused by the tumor.

**2.6 Autoimmune hepatitis**

differential diagnosis (Table 4).

liver diseases; nevertheless, it is not cancer specific. Comunale and colleagues (Comunale et al., 2010) identified more than 100 glycoproteins that are elevated in cirrhotic and HCC patients and that may serve as markers of the early development of HCC, and they

CRP and SAA levels are correlated with tumor activity (Jong, 2001), i.e., with increasing tumor load, necrotic tumors, and tissue destruction. Levels of these proteins are even increased by gastric and colorectal metastases. It has also been suggested that APPs provide an ideal environment for tumor recurrence or growth (Harimoto et al., 2009). It is also accepted that CRP-positivity in HCC patients often indicates a poor prognosis and portal vein invasion. As mentioned earlier in the chapter, hypoalbuminemia was considered a marker of liver impairment. For a long time, it was thought that tumor activity was responsible for the decrease in albumin level; however, recent studies (Al-Shaiba et al., 2004) have demonstrated that the reduction in serum albumin level is related to the inflammatory

Autoimmunity is also an important cause of liver disease. It encompasses a broad spectrum of diseases, including autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and autoimmune cholangitis. The most common are AIH and PBC. Clinical, biochemical, histopathological, and cholangiographic criteria are used for

AIH is characterized by severe liver damage with a modest or low elevation of alkaline phosphatase level (Oo, 2010). It is classed into three types according to the autoantibodies expressed: type 1, smooth muscle antibodies (SMA) or anti-nuclear antibodies (ANA) are present; type 2, anti-liver kidney microsomal antibodies (LKM) are present; and type 3, soluble liver antibodies (SLA) or liver/pancreas antigens are present. All of these antibodies induce proinflammatory cells that mostly activate T-helper cells, which cause liver damage.

Table 4. Comparative table for the differential diagnosis of autoimmune liver diseases. AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; AIC, autoimmune cholangitis; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase; Ig, immunoglobin; ANA, anti-nuclear antibodies; ASMA, anti-smooth muscle antibodies; LKM, anti-liver kidney microsomal; SLA, anti-soluble liver antigen; p-ANCA, perinuclear antineutrophil cytoplasmic antibodies; AMA, anti-mitochondrial antibodies; UDCA, ursodeoxycholic acid.

In PBC (Poupon, 2010), inflammation is associated with similar rates of apoptosis and proliferation of biliary cells. Cytolytic T cells are attracted to autoantibody-marked cells by cytokines and chemokines, which also induce the killing of cells via TNF-, CD-40, and Fas receptors. It is under this pressure that cholangiocytes proliferate to compensate for cell death. It has been suggested that the cholinergic pathway, the IGF-1 system, and estrogens (through alpha receptors) are possible mediators of this process.

Takahashi and colleagues (Takahasi et al., 2010) found that ALT and transferrin levels are correlated in autoimmune diseases and in other collagen-related diseases. CRP and AP levels are also correlated, at least in vasculitis syndrome. The explanation for these results is that, because the immunological response is increased in all these diseases, APPs are induced. IL-17 is a new cytokine that has been suggested to play an important role in the pathogenesis of AIH, as it is a strong neutrophil recruiter and, importantly, is also associated with the pathogenesis of ALD.

Furthermore, several authors have described "overlap syndromes". These entities are characterized by clinical or biochemical features common to two or more autoimmune diseases affecting the liver. In overlap syndromes, the magnitude of the immunological response in the early stage is elevated in that APP levels are altered to a greater extent. Comorbidities are not considered overlap syndromes. Kessel and colleagues (Kessel et al., 2007) reported that HCV-infected patients have a high titer of CRP antibodies, which is correlated with the level of rheumatoid factor, cryoglobulinemia, and the severity of liver disease, suggesting that it facilitates autoimmune liver disorders. This is important because it has been shown that 65% of patients infected with HCV have low titers of ANA, SMA, and anti-thyroid antibodies, and 7% have anti-LKM-1 antibodies. Some authors suggest that, if titers of these antibodies are >1:320, and/or hypergammaglobulinemia, and other risk factors (female gender, young age, and other autoimmune disorders) are present, it should be considered as a co-morbidity between HCV and AIH (Beuers, 2005).

#### **3. Clinical repercussions**

Clinically, insulin resistance (IR) and cardiovascular disease (CVD) have been suggested to arise from a common basis, i.e., chronic inflammation. As mentioned before, chronic liver diseases are important sources of a proinflammatory state and, therefore, several studies have tried to link liver impairment with the development of IR, type 2 diabetes mellitus (T2DM), and CVD. It is important to comment on this situation because several reports have suggested that the prevalence of chronic liver diseases will increase in the next few years and have shown that chronic liver diseases are currently among the leading causes of mortality in all parts of the world.

#### **3.1 Liver-related insulin resistance (IR)**

One of the manifestations of liver impairment is IR. IR is defined as an increased need for insulin in the peripheral tissues (muscle and adipose tissue) to achieve normal cellular glucose uptake and to reduce glucose output from the liver. The incidence of T2DM is increasing worldwide. IR is considered the pathophysiological basis of T2DM and is responsible for micro- and macrovascular complications associated with this condition. Some studies have associated IR with other deleterious effects on the biliary tract and liver such as the induction of gallstones and NAFLD, considered the most common chronic liver disease in Western countries and the liver manifestation of metabolic syndrome (MS), a pathology proven to be intrinsically related to IR. Some studies have even found that IR tends to favor the progression of NAFLD to NASH, cirrhosis, and HCC.

To comprehend the entire pathophysiology of IR, it is important to understand the normal physiology of insulin signaling. The main signaling pathway begins with the binding of insulin to its receptor, which activates insulin receptor substrates (ISR-1 or ISR-2), which activate phosphatidylinositol-3 kinase (PI3K). The subsequent cascade results in the production of protein kinase Corwhich promotes translocation of the glucose transporter, GLUT 4, and activation of Akt, facilitating glucose uptake, gluconeogenesis, and protein synthesis through the production of GSK-3, FOXO-1, and mTOR, respectively. The last-mentioned mediator also exerts negative feedback on insulin signaling by phosphorylating IRS-1 (Méndez et al., 2005) (figure 4).

Two main pathways are involved in liver-related IR. One path is through the production of free fatty acids, an increased level of which inhibits insulin-induced suppression of endogenous glucose production, and the other is through stimulation of gluconeogenesis, associated with activation of protein kinase C.

Nevertheless, there are other factors associated with IR, such as a mutation of 2,6 fructose bisphosphonatase that causes an alteration in the fructose 2,6-bisphosphonate, which causes three results: a lower efficiency of suppression of hepatic glucose production (increased gluconeogenesis); a disruption of glucose flux, and a decrease in insulin-induced Akt phosphorylation in the liver. Furthermore, phosphorylation of ISR-2 in the cascade of activation of insulin also has an important impact on glycogen synthesis. It has been demonstrated that ISR-2 phosphorylation inhibits glycogen synthetase-3-kinase, which, in normal situations, induces glycogen synthesis and increases the use of plasma glucose. In pathological cases, synthesis is interrupted and glucose is not taken up by cells, promoting the progression of IR.

Clinically, insulin resistance (IR) and cardiovascular disease (CVD) have been suggested to arise from a common basis, i.e., chronic inflammation. As mentioned before, chronic liver diseases are important sources of a proinflammatory state and, therefore, several studies have tried to link liver impairment with the development of IR, type 2 diabetes mellitus (T2DM), and CVD. It is important to comment on this situation because several reports have suggested that the prevalence of chronic liver diseases will increase in the next few years and have shown that chronic liver diseases are currently among the leading causes of

One of the manifestations of liver impairment is IR. IR is defined as an increased need for insulin in the peripheral tissues (muscle and adipose tissue) to achieve normal cellular glucose uptake and to reduce glucose output from the liver. The incidence of T2DM is increasing worldwide. IR is considered the pathophysiological basis of T2DM and is responsible for micro- and macrovascular complications associated with this condition. Some studies have associated IR with other deleterious effects on the biliary tract and liver such as the induction of gallstones and NAFLD, considered the most common chronic liver disease in Western countries and the liver manifestation of metabolic syndrome (MS), a pathology proven to be intrinsically related to IR. Some studies have even found that IR

To comprehend the entire pathophysiology of IR, it is important to understand the normal physiology of insulin signaling. The main signaling pathway begins with the binding of insulin to its receptor, which activates insulin receptor substrates (ISR-1 or ISR-2), which activate phosphatidylinositol-3 kinase (PI3K). The subsequent cascade results in the production of protein kinase Corwhich promotes translocation of the glucose transporter, GLUT 4, and activation of Akt, facilitating glucose uptake, gluconeogenesis, and protein synthesis through the production of GSK-3, FOXO-1, and mTOR, respectively. The last-mentioned mediator also exerts negative feedback on insulin signaling by

Two main pathways are involved in liver-related IR. One path is through the production of free fatty acids, an increased level of which inhibits insulin-induced suppression of endogenous glucose production, and the other is through stimulation of gluconeogenesis,

Nevertheless, there are other factors associated with IR, such as a mutation of 2,6 fructose bisphosphonatase that causes an alteration in the fructose 2,6-bisphosphonate, which causes three results: a lower efficiency of suppression of hepatic glucose production (increased gluconeogenesis); a disruption of glucose flux, and a decrease in insulin-induced Akt phosphorylation in the liver. Furthermore, phosphorylation of ISR-2 in the cascade of activation of insulin also has an important impact on glycogen synthesis. It has been demonstrated that ISR-2 phosphorylation inhibits glycogen synthetase-3-kinase, which, in normal situations, induces glycogen synthesis and increases the use of plasma glucose. In pathological cases, synthesis is interrupted and glucose is not taken up by cells, promoting

tends to favor the progression of NAFLD to NASH, cirrhosis, and HCC.

phosphorylating IRS-1 (Méndez et al., 2005) (figure 4).

associated with activation of protein kinase C.

the progression of IR.

**3. Clinical repercussions**

mortality in all parts of the world.

**3.1 Liver-related insulin resistance (IR)**

Fig. 4. Mechanisms involved in insulin signaling. Under normal circumstances, insulin binds to its receptor, which activates IRS 1/2, which then initiates a cascade of reactions that activate PKC, inducing the synthesis and migration of GLUT4 to the cell membrane, and Akt, which promotes glucose uptake, gluconeogenesis, and protein synthesis. mTOR also exerts negative feedback on the activation of IRS. IRS, insulin receptor substrate; PI3K, phosphatidylinositol-3 kinase; PKC, protein kinase C.

Fig. 5. Liver-related insulin resistance. FFA, free fatty acids; IRS, insulin receptor substrate.

As mentioned before, TNF- concentrations are elevated in liver diseases and, through the activation of inhibitory , which enables activation of nuclear factor-, cause upregulation of IL-6. IL-6 induces the suppressor of cytokine signaling (SOCS), which induces IR through a specific inhibitor of IRS-2 (SOCS-1) or by competition for the docking sites of SOCS-3 (Méndez et al., 2005).

Festa and colleagues (Festa et al., 2002) reported that APPs are not only impaired in cases of T2DM but proposed that they serve as early biomarkers of T2DM. CRP and fibrinogen were related to IR, but several studies have related them to adipose tissue secretion, and therefore to body mass index. Nevertheless, they also found that PAI-1 was independently related to the development of T2DM when compared with BMI and other risk factors, even IR, suggesting that this APP is suitable for early screening of people who are predisposed to T2DM.

Recent studies have also confirmed a close relationship between IR and liver diseases such as chronic hepatitis caused by HBV and HCV, hemochromatosis, cirrhosis, and HCC. Some authors have even reported so-called "hepatogenous diabetes", which is recognized by the World Health Organization as an independent entity that refers to the development of the T2DM due to cirrhosis.

It is important to note that the incidence of viral hepatitis has increased worldwide, especially the types that produce a chronic state. In regards to these, some studies have reported that HCV infection induces degradation of IRS-1 through activation of the mTOR pathway (genotype 1) or SOCS-7 and PPAR- (genotype 3). In either eventuality, the result is IR. It is also important to mention that these two genotypes have been related to IR in HCV infection.

Several studies have been performed to analyze the relationships of IR and T2DM with liver disease. Although the prevalence of liver diseases varies between countries, it has been noted that liver cirrhosis and viral hepatitis C infection have the strongest relationship with T2DM; nevertheless, other liver diseases have also been correlated with T2DM. Table 5 shows the prevalence of these diseases.

#### **3.2 Relationship between cardiovascular risk and liver impairment**

Metabolic syndrome is the pandemic of the new millennium and is considered a major risk factor for CVD. NAFLD is now considered a liver manifestation of this syndrome. As mentioned before, IR is the pathophysiological basis of metabolic syndrome and is therefore closely linked to both CVD and liver diseases.

As also mentioned above, CRP is considered the most important APP correlated with IR and CVD; nevertheless, there are some studies that have shown that CRP has anti-inflammatory properties and is involved in the reduction of the development of atherosclerosis in mouse models with hypercholesterolemia. This is most probably due to CRP-mediated upregulation of the IL-1 receptor antagonist and upregulation of serum leptin-interacting protein activity.

NASH, as also mentioned before, characteristically elevates CRP, PAI-1, and fibrinogen activities (more so than NAFLD), which are also correlated with CVD and could be considered markers for this entity because these APPs have been demonstrated to be related to cardiovascular events independently of other risk factors such as age, visceral adiposity, and metabolic abnormalities. As such, patients with viral hepatitis also have a markedly greater carotid artery intimae medial thickness. Figure 6 shows the physiopathology of this interaction.

upregulation of IL-6. IL-6 induces the suppressor of cytokine signaling (SOCS), which induces IR through a specific inhibitor of IRS-2 (SOCS-1) or by competition for the docking

Festa and colleagues (Festa et al., 2002) reported that APPs are not only impaired in cases of T2DM but proposed that they serve as early biomarkers of T2DM. CRP and fibrinogen were related to IR, but several studies have related them to adipose tissue secretion, and therefore to body mass index. Nevertheless, they also found that PAI-1 was independently related to the development of T2DM when compared with BMI and other risk factors, even IR, suggesting that this APP is suitable for early screening of people who are predisposed to

Recent studies have also confirmed a close relationship between IR and liver diseases such as chronic hepatitis caused by HBV and HCV, hemochromatosis, cirrhosis, and HCC. Some authors have even reported so-called "hepatogenous diabetes", which is recognized by the World Health Organization as an independent entity that refers to the development of the

It is important to note that the incidence of viral hepatitis has increased worldwide, especially the types that produce a chronic state. In regards to these, some studies have reported that HCV infection induces degradation of IRS-1 through activation of the mTOR pathway (genotype 1) or SOCS-7 and PPAR- (genotype 3). In either eventuality, the result is IR. It is also important to mention that these two genotypes have been related to IR in

Several studies have been performed to analyze the relationships of IR and T2DM with liver disease. Although the prevalence of liver diseases varies between countries, it has been noted that liver cirrhosis and viral hepatitis C infection have the strongest relationship with T2DM; nevertheless, other liver diseases have also been correlated with T2DM. Table 5

Metabolic syndrome is the pandemic of the new millennium and is considered a major risk factor for CVD. NAFLD is now considered a liver manifestation of this syndrome. As mentioned before, IR is the pathophysiological basis of metabolic syndrome and is therefore

As also mentioned above, CRP is considered the most important APP correlated with IR and CVD; nevertheless, there are some studies that have shown that CRP has anti-inflammatory properties and is involved in the reduction of the development of atherosclerosis in mouse models with hypercholesterolemia. This is most probably due to CRP-mediated upregulation of the IL-1 receptor antagonist and upregulation of serum leptin-interacting

NASH, as also mentioned before, characteristically elevates CRP, PAI-1, and fibrinogen activities (more so than NAFLD), which are also correlated with CVD and could be considered markers for this entity because these APPs have been demonstrated to be related to cardiovascular events independently of other risk factors such as age, visceral adiposity, and metabolic abnormalities. As such, patients with viral hepatitis also have a markedly greater carotid artery intimae medial thickness. Figure 6 shows the physiopathology of this

**3.2 Relationship between cardiovascular risk and liver impairment**

sites of SOCS-3 (Méndez et al., 2005).

T2DM.

T2DM due to cirrhosis.

shows the prevalence of these diseases.

closely linked to both CVD and liver diseases.

HCV infection.

protein activity.

interaction.

Table 5. Prevalence of type 2 diabetes mellitus (T2DM) and liver diseases reported from several studies worldwide (from Kobashi et al., 2010).

Fig. 6. Relationship between liver steatosis and cardiovascular events (atherosclerosis). Several APPs are elevated in NASH, which may directly injure the endothelium, but adipose tissue alone may also produce the same alteration. FFA, free fatty acids; IR, insulin resistance; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; TG, triglyceride; VLDL, very low density lipoprotein; HDL, high density lipoprotein.

It is important to mention that several risk factors for the development of CVD are also important risk factors for diabetes and some liver diseases. The transport of cholesterol is intrinsically related to these pathologies. Recently, studies have focused on the ATP binding cassette, A-1 (ABCA-1), an ATP-binding membrane transporter that plays an important role in cholesterol efflux from tissues and interacts directly with apolipoprotein A1, which is a structural component of high density lipoprotein cholesterol (HDL-c) and therefore plays a crucial role in lipid transportation. Because ABCA-1 and Apo-A1 have been demonstrated to be reduced in some liver diseases, it is comprehensible that liver impairment could be reflected in decreased HDL-c levels through this mechanism (Figure 7). HDL-c plays a protective role in cardiovascular events because it transports cholesterol from peripheral tissues to the liver where lipid is metabolized. The metabolism and transport of cholesterol demonstrates the close relationship between liver diseases and cardiovascular events.

Several studies have also pointed out that adiponectin, an adipocyte-derived enzyme, plays a protective role in the pathogenesis of liver impairment (induction of NAFLD) and therefore, in the increase in cardiovascular risk. Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are important rate-limiting enzymes for fatty acid synthesis. The

Fig. 7. Cholesterol reverse transport metabolism. Oxysterols (ligands for LXR receptors) and activators of PPAR induce the production of ABC-A1, which transports molecules of cholesterol from inside the cell to the outer cell membrane. Once presented, the structural molecule, ApoA1, of HDL-c attaches to the molecules of cholesterol and they are transported to the liver where they can be metabolized and excreted from the organism. In the absence of HDL-c stimulation (i.e., in liver diseases), the LXR- receptor and RXR are activated and inhibit ABC-A1, maintaining cholesterol accumulation within tissues. LXR, liver X receptor; RXR, retinoid X receptor.

activity of these enzymes and that of carnitine palmitoyl transferase I (CPT-I), which mediates fatty acid entry to the mitochondrion, where the fatty acids are degraded, are regulated by adiponectin. This enzyme promotes CPT-I expression and inhibits ACC and FAS expression, reducing serum and hepatic lipid concentrations and, thus, the activity of TNF-. Uribe and colleagues (Uribe et al., 2008) have shown that adiponectin expression is reduced in metabolic liver diseases and in obesity.

#### **4. Conclusions**

120 Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases

Fig. 6. Relationship between liver steatosis and cardiovascular events (atherosclerosis). Several APPs are elevated in NASH, which may directly injure the endothelium, but adipose tissue alone may also produce the same alteration. FFA, free fatty acids; IR, insulin resistance; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; TG, triglyceride; VLDL, very low density lipoprotein; HDL, high density lipoprotein.

It is important to mention that several risk factors for the development of CVD are also important risk factors for diabetes and some liver diseases. The transport of cholesterol is intrinsically related to these pathologies. Recently, studies have focused on the ATP binding cassette, A-1 (ABCA-1), an ATP-binding membrane transporter that plays an important role in cholesterol efflux from tissues and interacts directly with apolipoprotein A1, which is a structural component of high density lipoprotein cholesterol (HDL-c) and therefore plays a crucial role in lipid transportation. Because ABCA-1 and Apo-A1 have been demonstrated to be reduced in some liver diseases, it is comprehensible that liver impairment could be reflected in decreased HDL-c levels through this mechanism (Figure 7). HDL-c plays a protective role in cardiovascular events because it transports cholesterol from peripheral tissues to the liver where lipid is metabolized. The metabolism and transport of cholesterol demonstrates the close relationship between liver diseases and cardiovascular events. Several studies have also pointed out that adiponectin, an adipocyte-derived enzyme, plays a protective role in the pathogenesis of liver impairment (induction of NAFLD) and therefore, in the increase in cardiovascular risk. Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are important rate-limiting enzymes for fatty acid synthesis. The

Although albumin level is generally correlated with liver disease, it is not specific for the various liver diseases. Consequently, several studies have been conducted to identify biomarkers specific for each liver disease. Table 6 lists APPs associated with various liver diseases.

It is important to mention that although chemical analysis has improved over the years, the clinical status of the patient is the most important and reliable factor in the approach of the patient. APPs measurement gives the clinician, the proper guidance and orientation of the problems, that usually are subclinical, but they are not the gold standard for any liver disease; therefore, they all need a confirmation with some other studies.

Table 6. APP, acute-phase protein; AFP, alpha-fetoprotein; LBP-1, lipopolysaccharide binding protein 1; NAFLD, nonalcoholic fatty liver disease; ALT, alanine aminotransferase; NASH, nonalcoholic steatohepatitis; GGT, gamma-glutamyl transpeptidase; CRP, c-reactive protein; SAA, serum amyloid A; PTX3, pentrexin 3; A2MG, alpha-2 macroglobulin; ApoA1, apolipoprotein A1; VDBP, vitamin D binding protein; HA, hyaluronic acid; IGF-1, insulinlike growth factor-1; HCC, hepatocellular carcinoma; A1AT, alpha-1 antitrypsin; AP, alkaline phosphatase.

#### **5. References**

**Liver disease Positive APP Negative APP**

**Viral hepatitis** CRP, SAA, Fibrinogen,

**Cirrhosis** A2MG ApoA1, VDBP, HA, IGF-1

Table 6. APP, acute-phase protein; AFP, alpha-fetoprotein; LBP-1, lipopolysaccharide binding protein 1; NAFLD, nonalcoholic fatty liver disease; ALT, alanine aminotransferase; NASH, nonalcoholic steatohepatitis; GGT, gamma-glutamyl transpeptidase; CRP, c-reactive protein; SAA, serum amyloid A; PTX3, pentrexin 3; A2MG, alpha-2 macroglobulin; ApoA1, apolipoprotein A1; VDBP, vitamin D binding protein; HA, hyaluronic acid; IGF-1, insulinlike growth factor-1; HCC, hepatocellular carcinoma; A1AT, alpha-1 antitrypsin; AP,

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### **Acute Phase Proteins as Biomarkers of Disease: From Bench to Clinical Practice**

Francisco J. Salgado, Pilar Arias, Ana Canda-Sánchez and Montserrat Nogueira *University of Santiago de Compostela Spain*

#### **1. Introduction**

126 Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases

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The term acute phase response (APR) refers to the inflammatory response of the host occurring shortly after the tissue injury. It comprises a wide variety of reactions started by different causes, like infection, tissue injury, burn, trauma, surgery, cancer or immunological disorders. These reactions aim to prevent ongoing tissue damage, isolate and eliminate the cause of the inflammation, and begin the repair process necessary to restore the normal function. Usually, the local response is accompanied by a systemic reaction characterized by the fast alteration of the concentrations of several plasmatic proteins, the APPs (Acute Phase Proteins) produced by the liver (Baumann & Gauldie, 1994). In some diseases, the persistent immunological activation can cause chronic inflammation, often with pathological consequences. In other words, APR is a physiological condition occurring at the beginning of the inflammatory process and it is independent of the inflammation origin.

#### **1.1 The inflammatory response**

The inflammation is the ordered process mediated by the appearance of intercellular adhesion molecules on endothelia, and various inflammatory mediators released by tissue cells and leucocytes in response to tissue aggression; it is a protective response to injury or destruction of tissues, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. It is the first reaction of the body under a situation of immunological stress: the innate non-specific response preceding specific immune reactions.

The term inflammation is purely descriptive and originally defined by the four Latin words dolor, rubor, calor and tumor (meaning pain, redness, warmth and swelling); these are the result of changes in the local blood vessels which in turn, lead to their dilatation and increased stickiness and permeability for passing leukocytes. Combined, the cell and fluid leakage into the tissue, as well as their local activities, account for the pain and swelling. The function of this inflammatory response is to bring serum molecules and immune cells to the damaged zone. In this response three principal components can be considered: i) increased blood supply, ii) increased capillary permeability and, iii) migration of cells out of the vessels and into the tissues.

In an inflammatory reaction there are implicated several cells of the immunological system and a great variety of response mediators. The uncontrolled migration of different leukocytes populations would make impossible the generation and control of the immune response. In this setting, the endothelium plays a key role. Endothelial cells are multifunctional cells that form a thin layer in the interior surface of blood vessels (Cines et al., 1998). They are responsible for maintaining the vascular homeostasis but, also, they are capable of secreting biologically active mediators when affected by infection, stress, hypertension, dyslipidemia or high homocysteine levels. The primary essential function of these endothelial cells is to regulate the permeability of the blood vessel and the exchange of fluids and cells between the blood and the surrounding tissue. In the inflammatory response they raise the expression of cell adhesion molecules that, therefore, increments the number of leukocytes binding endothelium (Pate et al., 2010). The increased permeability of the endothelium allows the migration of these cells, a process called extravasation, where they will exert its function. That is why they are so important for the recruitment and orchestration of an acute inflammatory response.

The role of the different cells and different interactions has been extensively described (Imhof & Aurrand-Lions, 2004; Langer & Chavakis, 2009; Ley et al., 2007; Schenkel et al., 2004; Vestweber, 2007), but briefly we are going to comment those. Normally, neutrophils are the first cells to appear at acute phase response, followed by macrophages (monocytes), lymphocytes (if there is an immunological challenge) and a small amount of basophils and eosinophils; if an extreme vascular leakage occurs, red blood cells may also be found. We can distinguish three steps for the migration of cell to the tissue during the inflammatory response (Butcher, 1991):

1-Receptor-mediated recruitment and adhesion to the endothelial cells: the regulation of the expression of selectin-P and selectin-E by the endothelium is key in this process (Lasky, 1995; Lawrence & Springer, 1991). The expression of P-selectin is increased by inflammatory molecules such as histamine and thrombine, it is a weak binder to the leukocytes but causes them to "slow" their movement in the vessel. Selectin-E expression, on the other hand, it is upregulated by cytokines produced by injured cells, such as IL-1 and TNF-; these cytokines also activate and increase the expression of integrins on the circulating leukocytes. Activated leukocytes, therefore, increase the affinity of their integrins for certain ligands on the endothelial surface, attaching them firmly to the endothelium.

2- Transmigration, or the movement of the activated leukocytes through the endothelium to reach the damaged tissue: the leukocytes adhered to the endothelium are stimulated by the chemokine gradient to move between the endothelial cells and across the basal membrane to the damaged area. Among the chemotactic factors we can find: i) products of the complement like C3a, C4a, C5a (Hartmann et al., 1997; Okusawa et al., 1988), ii) fibrin degradation products (FDPs) like the D-dimer (Gross et al., 1997), iii) prostaglandins (PGD2, PGE2) (Konya et al., 2010; Kuehn et al., 2011), iv) leukotrienes (LTB4) (Aked & Foster, 1987) and chemokines like CCL5 (Kawai et al., 1999; Murooka et al., 2008) and IL-8 (Huber et al., 1991).

3- Movement of the activated leukocytes through the tissue: the binding to extracellular matrix proteins through the integrins and the CD44 protein allows these cells to arrive to the origin of the inflammation following the chemokine gradient (Kinashi, 2005).

#### **1.1.1 The Pro-inflammatory cytokines**

But in this local inflammatory process, activated leukocytes are not passive: in turn, they release cytokines and other mediators like glucocorticoids into the bloodstream, thus eliciting a widespread reaction in the organism. There are at least 15 cytokines known to be involved in this process. Some of them are positive/negative regulators of cell growth (IL-2, IL-3, IL-4, IL-7, IL-10, IL-11, IL-12 and GM-CSF), while others have got pro-inflammatory (TNF-α/β, IL-1α/β, IL-6, IFN-α/γ, IL-8) or anti-inflammatory activities (IL-1 antagonists, soluble IL-1R, IL-1 and TNF-α binding protein)(van Miert, 1995).

Proinflammatory cytokines, released mainly by activated macrophages, are crucial for the induction of other cytokines (IL-6, IL-8), while agents such as platelet activating factor, prostaglandins, leukotrienes and nitric oxide increase the chemotactic gradient and therefore the leukocyte recruitment to the inflammation site. They are also responsible for the fever and catabolism of muscle proteins through their action at the CNS level: We will briefly comment some carachteristics of the proinflammatory cytokines involved in the acute phase reaction and the production of APPs:

TNF-α is the main cytokine that mediates acute inflammation. TNF is produced by monocytes, macrophages, dendritic cells, Th1 cells, and other cells. Some of its functions include: to stimulate the cells from the endothelium to produce selectins and the leukocytes to express integrins, to activate the coagulation pathway, to increase the production of chemokines in endothelial cells and macrophages, to activate neutrophils and their killer activity, etc (Baud & Karin, 2001). It is also responsible for stimulating the liver to produce APPs (Xanthoulea et al., 2004), and acting on muscles (Collins & Grounds, 2001) and fat (Lu et al., 2006; Plomgaard et al., 2008; Rydén et al., 2002) to stimulate catabolism for energy conversion. In addition, it interacts with the hypothalamus to induce lack of appetite (Tracey et al., 1990), fever (Rothwell & Hopkins, 1995; Stefferl et al., 1996) and sleep (Deboer et al., 2002; Fang et al., 1997). It also participates in the cicatrization process through the stimulation of collagen and collagenase synthesis (Theiss et al., 2005).

128 Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases

leukocytes populations would make impossible the generation and control of the immune response. In this setting, the endothelium plays a key role. Endothelial cells are multifunctional cells that form a thin layer in the interior surface of blood vessels (Cines et al., 1998). They are responsible for maintaining the vascular homeostasis but, also, they are capable of secreting biologically active mediators when affected by infection, stress, hypertension, dyslipidemia or high homocysteine levels. The primary essential function of these endothelial cells is to regulate the permeability of the blood vessel and the exchange of fluids and cells between the blood and the surrounding tissue. In the inflammatory response they raise the expression of cell adhesion molecules that, therefore, increments the number of leukocytes binding endothelium (Pate et al., 2010). The increased permeability of the endothelium allows the migration of these cells, a process called extravasation, where they will exert its function. That is why they are so important for the recruitment and

The role of the different cells and different interactions has been extensively described (Imhof & Aurrand-Lions, 2004; Langer & Chavakis, 2009; Ley et al., 2007; Schenkel et al., 2004; Vestweber, 2007), but briefly we are going to comment those. Normally, neutrophils are the first cells to appear at acute phase response, followed by macrophages (monocytes), lymphocytes (if there is an immunological challenge) and a small amount of basophils and eosinophils; if an extreme vascular leakage occurs, red blood cells may also be found. We can distinguish three steps for the migration of cell to the tissue during the inflammatory

1-Receptor-mediated recruitment and adhesion to the endothelial cells: the regulation of the expression of selectin-P and selectin-E by the endothelium is key in this process (Lasky, 1995; Lawrence & Springer, 1991). The expression of P-selectin is increased by inflammatory molecules such as histamine and thrombine, it is a weak binder to the leukocytes but causes them to "slow" their movement in the vessel. Selectin-E expression, on the other hand, it is upregulated by cytokines produced by injured cells, such as IL-1 and TNF-; these cytokines also activate and increase the expression of integrins on the circulating leukocytes. Activated leukocytes, therefore, increase the affinity of their integrins for certain ligands on the

2- Transmigration, or the movement of the activated leukocytes through the endothelium to reach the damaged tissue: the leukocytes adhered to the endothelium are stimulated by the chemokine gradient to move between the endothelial cells and across the basal membrane to the damaged area. Among the chemotactic factors we can find: i) products of the complement like C3a, C4a, C5a (Hartmann et al., 1997; Okusawa et al., 1988), ii) fibrin degradation products (FDPs) like the D-dimer (Gross et al., 1997), iii) prostaglandins (PGD2, PGE2) (Konya et al., 2010; Kuehn et al., 2011), iv) leukotrienes (LTB4) (Aked & Foster, 1987) and chemokines like CCL5 (Kawai et al., 1999; Murooka et al., 2008) and IL-8 (Huber et al.,

3- Movement of the activated leukocytes through the tissue: the binding to extracellular matrix proteins through the integrins and the CD44 protein allows these cells to arrive to the

But in this local inflammatory process, activated leukocytes are not passive: in turn, they release cytokines and other mediators like glucocorticoids into the bloodstream, thus eliciting a widespread reaction in the organism. There are at least 15 cytokines known to be

origin of the inflammation following the chemokine gradient (Kinashi, 2005).

orchestration of an acute inflammatory response.

endothelial surface, attaching them firmly to the endothelium.

**1.1.1 The Pro-inflammatory cytokines**

response (Butcher, 1991):

1991).

IL-1 function is similar to TNF- as it also mediates acute inflammatory responses, and together they enhance the inflammatory response (Dinarello & Wolff, 1993; Vassalli, 1992). Monocytes, macrophages, dendritic cells and endothelial cells are the main sources of IL-1. Functions of IL-1 include the promotion of the inflammation through the activation of the coagulation pathway and the synthesis of adhesion factors on endothelial cells and leukocytes (Bevilacqua et al., 1984; Nawroth et al., 1986) and the activation of macrophages (Dinarello, 1988). It is also responsible for stimulating the liver to produce APPs (Baumann & Gauldie, 1994; Mortensen et al., 1988; Prowse & Baumann, 1989), for activating the catabolism of the fat tissue for energy conversion (Feingold et al., 1992; Tocco-Bradley et al., 1987); and for regulating the synthesis of collagen and collagenase for scar tissue formation (Duncan & Berman, 1989; Mizel et al., 1981).

It is also noteworthy the role that IL-1 plays in the induction of the fever and sleep (Krueger et al., 1984; Opp & Krueger, 1991), as well as the stimulatory effect of IL-1 on the pituitaryadrenal axis in the CNS (Besedovsky et al., 1986; Sapolsky et al., 1987), thus making it responsible for the control of food intake (Luheshi et al., 1999) during the acute phase response.

Interleukin-1 and TNF- have also being linked to the depression and sickness behaviour associated to the inflammatory response (Dantzer, 2001; Konsman et al., 2002). One theory behind this effect it is the role of IL-1 over serotoninergic neurons, where IL-1 could activate the serotonin transporter directly. This could lead to the removal of serotonin from the synaptic cleft, thus generating a serotoninergic deprivation signal together with the tryptophan deprivation derived from the APPs production (Leonard & Song, 1999).

IL-6 stimulates the liver to produce APPs and it is released by many cells including Tlymphocytes, macrophages, monocytes, endothelial cells, and fibroblasts (Prowse & Baumann, 1989). It is also responsible for the proliferation stimulation of B-lymphocytes (Muraguchi et al., 1988; Taga et al., 1987) and increases the neutrophil activity (Brom & König, 1992).

The outcome of the inflammation will depend on the tissue as well as the extent of injury and the injurious agent that has caused it. Usually, the resolution will bring the inflamed tissue to its status prior to the injury but, some times, the inflammation is not resolved properly. Sometimes a persistent damage or an incomplete resolution process can produce a chronic inflammation due to the continuous production of pro-inflammatory cytokines. Often, the reason behind the abnormal inflammatory response is not known, and it is the reason behind many human diseases. Some of them have got an immunological origin like the allergic reactions or myopathies (Lundberg & Grundtman, 2008); but sometimes the origin is non-immunological like in rheumatoid arthritis, cancer (Coussens & Werb, 2002), atherosclerosis (Libby, 2002), ischaemic heart disease (Maseri et al., 1996), inflammatory bowel diseases (Fiocchi, 1998), etc (many articles, reviews and books cover these topics, so this is not an extensive list). In all of the above mentioned disorders we can also see alterations in the levels of different APPs produced by the liver; the next section will present an extensive list of these proteins, where the main characteristics of the different groups will be commented briefly.

#### **2. Acute phase proteins**

APPs is a group of plasma proteins whose concentration varies in response to inflammation (Baumann & Gauldie, 1994). They are synthesized mainly by the liver but also by other cell types (monocytes, endothelial cells, fibroblasts and adipocytes). According to the variations in the concentration of these proteins they can be classified in positive APPs, those that increase the concentration, and negative APPs, those that show decreased concentrations upon the inflammatory response. There are also other sub-classifications for the positive APPs, according to the variation in concentration: Group I positive APPs increase up to 50% (ceruloplasmin and complement factor-3 (C3)), Group II from 2 to 5 times (haptoglobin, fibrinogen, α-globulins with antiprotease-activity and lipopolysaccharide binding protein), and Group III from 5 to more than 1000 times (CRP and SAA) (Dowton & Colten, 1988).

There is a lot of diversity regarding the timings that each of these proteins show any variation on its levels. Some proteins alter their levels as soon as 4 h, while others can take up to 24 h or even days. CRP (C-reactive protein) and SAA (serum amyloid A) are some of these very rapidly increasing proteins: they elevate their levels within the first 4 h after tissue injury, opposite to the lipopolysaccharide binding protein, that requires at least 8 h, or the serum alpha-l-acid glycoprotein, that needs 48 h. Some of these proteins remain elevated for various days, while other only for few hours. The origin behind the inflammation is also important: the plasma fibrinogen shows a peak in the concentration at 24 h-48 h or at 96 h, depending on the inflammatory stimulus (Colley et al., 1983; Gruys et al., 2005)

As commented before TNF-, IL-1, and IL-6 are the main cytokines involved in the hepatocytic secretion of APPs, but there are many others. Indeed, all type of combinatorial effects can regulate the hepatocytic secretion of an APP (addition, synergy, antagonism). IL-1 and IL-6 have been used to classify APPs into two subgroups: Type 1 requires the

IL-6 stimulates the liver to produce APPs and it is released by many cells including Tlymphocytes, macrophages, monocytes, endothelial cells, and fibroblasts (Prowse & Baumann, 1989). It is also responsible for the proliferation stimulation of B-lymphocytes (Muraguchi et al., 1988; Taga et al., 1987) and increases the neutrophil activity (Brom &

The outcome of the inflammation will depend on the tissue as well as the extent of injury and the injurious agent that has caused it. Usually, the resolution will bring the inflamed tissue to its status prior to the injury but, some times, the inflammation is not resolved properly. Sometimes a persistent damage or an incomplete resolution process can produce a chronic inflammation due to the continuous production of pro-inflammatory cytokines. Often, the reason behind the abnormal inflammatory response is not known, and it is the reason behind many human diseases. Some of them have got an immunological origin like the allergic reactions or myopathies (Lundberg & Grundtman, 2008); but sometimes the origin is non-immunological like in rheumatoid arthritis, cancer (Coussens & Werb, 2002), atherosclerosis (Libby, 2002), ischaemic heart disease (Maseri et al., 1996), inflammatory bowel diseases (Fiocchi, 1998), etc (many articles, reviews and books cover these topics, so this is not an extensive list). In all of the above mentioned disorders we can also see alterations in the levels of different APPs produced by the liver; the next section will present an extensive list of these proteins, where the main characteristics of the different groups will

APPs is a group of plasma proteins whose concentration varies in response to inflammation (Baumann & Gauldie, 1994). They are synthesized mainly by the liver but also by other cell types (monocytes, endothelial cells, fibroblasts and adipocytes). According to the variations in the concentration of these proteins they can be classified in positive APPs, those that increase the concentration, and negative APPs, those that show decreased concentrations upon the inflammatory response. There are also other sub-classifications for the positive APPs, according to the variation in concentration: Group I positive APPs increase up to 50% (ceruloplasmin and complement factor-3 (C3)), Group II from 2 to 5 times (haptoglobin, fibrinogen, α-globulins with antiprotease-activity and lipopolysaccharide binding protein), and Group III from 5 to more than 1000 times (CRP and SAA) (Dowton & Colten, 1988). There is a lot of diversity regarding the timings that each of these proteins show any variation on its levels. Some proteins alter their levels as soon as 4 h, while others can take up to 24 h or even days. CRP (C-reactive protein) and SAA (serum amyloid A) are some of these very rapidly increasing proteins: they elevate their levels within the first 4 h after tissue injury, opposite to the lipopolysaccharide binding protein, that requires at least 8 h, or the serum alpha-l-acid glycoprotein, that needs 48 h. Some of these proteins remain elevated for various days, while other only for few hours. The origin behind the inflammation is also important: the plasma fibrinogen shows a peak in the concentration at 24 h-48 h or at 96 h,

depending on the inflammatory stimulus (Colley et al., 1983; Gruys et al., 2005)

As commented before TNF-, IL-1, and IL-6 are the main cytokines involved in the hepatocytic secretion of APPs, but there are many others. Indeed, all type of combinatorial effects can regulate the hepatocytic secretion of an APP (addition, synergy, antagonism). IL-1 and IL-6 have been used to classify APPs into two subgroups: Type 1 requires the

be commented briefly.

**2. Acute phase proteins**

König, 1992).

synergistic effect of both interleukins for maximum synthesis (examples: CRP, SAA, Alpha-1-acid glycoprotein 1); type 2 are those that only require IL-6 and usually IL-1 acts suppressing more than enhancing its production (examples: fibrinogen chains, haptoglobin, alpha-2-Macroglobulin (Ramadori & Christ, 1999)). There are some cytokines that regulate the expression of one or two APPs, like Activin A, a cytokine related with the superfamily of TGF-β. On the other hand, other APPs are the result of a wider combination of cytokines. That is the case of SAA that requires the synergitic effect of bacterial LPS and several cytokines (mainly IL-1, IL-6, and TNF, but also LIF, CNTF, oncostatin M, IL-11, and cardiotrophin-1) to be produced (Benigni et al., 1996).

The main structural characteristics of APPs can be found in Table 1. Regarding their functions, summarized in Table 2, they are opsonisation and elimination of microorganisms and their products (CPR), activation of the complement system (complement C4a, C4b) and the lectin complement pathway (manan-binding lectin), inhibition of certain enzymes (alpha-1-antitrypsin, alpha-2-antiplasmin) or interleukins (interleukin-1 receptor antagonist protein), blockade of free radicals and/or elimination of haemoglobin residues (haptoglobin, serotransferrin, hemopexin), regulation of the immune response (serum amyloid P), coagulation and tissue repair/remodelling (fibrinogen, plasminogen), transport of ions (apolipoproteins, calcitonin precursor) and hormones (serum albumin), etc. Others, like alpha 2-macroglobulin and the coagulation factors, also play an important role in wound healing, because they collaborate with the immune system by increasing the vascular permeability, because they have chemotactic properties, or even because they are able of trapping pathogens in local blood clots.

#### **3. Acute phase proteins currently used in routine clinical practice**

As compared with cytokine and other short distance mediators of cellular responses production, which occurs in pulsed patterns cleared from the circulation within a few hours due to their small size, the native molecular mass of APPs secreted by the liver is larger than the kidney filtration cutoff (about 45 kDa). An example is albumin, which is just larger and has a lifetime of about 21 days. This fact, together with their large half-life, ensures an extended residence time in plasma, which means that levels of some APPs may remain unchanged for days and may be useful for diagnostic purposes. Currently, alterations in levels of some APPs are used to assess either the health/nutritional status of an individual or, mainly, to check for the presence of reactive processes, which has more advantages than measuring IL-6, a sensitive indicator of inflammation or infection but with a normal range of 0-5 pg/ml and short plasma clearing times. An analysis of US Food and Drug Administration (FDA) approvals for IVD assays aimed to the quantitative detection of acute phase reactants reveals about 80 companies involved (Table 3), of which 15 dominate the market: Abbott Laboratories, Beckman Coulter, Dade Behring, Diasorin, Globalemed, Kamiya Biomedical, Kent Laboratories, Nitto Boseki, Olympus Life Science Research, Ortho-Clinical Diagnostics, Polymedco, Randox Laboratories, Roche Diagnostics, Siemens Medical Solutions Diagnostics, and The Binding Site.

About 51% of FDA-cleared or approved IVD assays are aimed to the detection of unique plasma or serum proteins that carry out their normal function in plasma. Most of these proteins are negative or positive acute phase reactants, like albumin, C-reactive protein, 1 antitrypsin, transferrin, ceruloplasmin, or fibrinogen. Table 4 summarizes different tests from the companies listed in Table 3 intended to the detection of APPs in clinical practice.

132 Acute Phase Proteins as Early Non-Specific Biomarkers of Human and Veterinary Diseases Acute Phase Proteins as Biomarkers of Disease: From Bench to Clinical Practice 133

Table 1. Main characteristics of APPs. There are represented the classical APPs and other proteins that have been reported as inflammation related proteins, and cited as putative APPs. Abbreviatures: Alternative Sequence, Alt-Seq; Disulfide Bond, -S-S-; Immunoglobulin, Ig; Interleukin, IL; Major Histocompatibility Complex, MHC; Modified residue, Mod-Res; Natural variants, Nat-Var; Phosphoserine, PSer; Phosphothreonine, PThr; Phosphotyrosine, PTyr; other abbreviatures are specified in the text.

Table 1. Main characteristics of APPs. There are represented the classical APPs and other proteins that have been reported as inflammation related proteins, and cited as putative

Immunoglobulin, Ig; Interleukin, IL; Major Histocompatibility Complex, MHC; Modified residue, Mod-Res; Natural variants, Nat-Var; Phosphoserine, PSer; Phosphothreonine, PThr;

APPs. Abbreviatures: Alternative Sequence, Alt-Seq; Disulfide Bond, -S-S-;

Phosphotyrosine, PTyr; other abbreviatures are specified in the text.

Table 2. Main functions of Acute Phase Proteins.

Existing APPs test provide a spectrum of clinical information, including definitive diagnosis of acute events (e.g. CRP), prediction of disease risk (CRP increases in coronary disease) or detection of disease recurrence. Thus, if we check the clinical diagnostic catalog at the Beckman Coulter home page, one of the companies with the broader list of IVD tests for APPs (Table 3), we will find that those tests have the following intended uses https://www.beckmancoulter.com/eCatalog/Catalog/Disease\_Management): albumin, to evaluate the hepatic/renal function, infectious diseases, inflammatory responses or nutritional assesment; alpha 1 acid glycoprotein, to assess the existence of infectious diseases or inflammation; alpha 1 antitrypsin, to check the presence of infectious diseases or inflammation; alpha 2 macroglobulin and antithrombin III, to detect anemia, thrombofilia or to assay the hepatic function; ApoA1 and ApoB, to measure the cardiovascular risk; ceruloplasmin, for the evaluation of the hepatic function or the detection of infection/inflammation; complement factors (C3 and C4), to evaluate the liver function or unmask autoimmune or inflammatory processes; fibrinogen, for thrombofilia and bledding disorders detection; haptoglobin, to uncover infectious diseases, inflammation, and anemia; plasminogen, to assess fibrinolysis; transferrin, to value the presence of anemia, the nutritional status, the presence of infectious diseases, or the renal function; and finally transthyretin/prealbumin, for hepatic function and nutritional assesment. In addition, detection of beta 2 microglobulin (e.g. Quantikine® IVD® Human 2M Immunoassay, R&D) is an aid in the diagnosis of autoimmune diseases (rheumatoid arthritis, systemic lupus erithematosus), viral infections or reduced glomerular filtration rates (kidney diseases). On the other hand, low levels of plasminogen activator inhibitor 1 (PAI-1) are linked to bleeding, while its elevation is related with an increased number of blood clots (surgery, infection, diabetes) and elevated risk of heart attack or coronary artery disease (CAD).

Existing APPs test provide a spectrum of clinical information, including definitive diagnosis of acute events (e.g. CRP), prediction of disease risk (CRP increases in coronary disease) or detection of disease recurrence. Thus, if we check the clinical diagnostic catalog at the Beckman Coulter home page, one of the companies with the broader list of IVD tests for APPs (Table 3), we will find that those tests have the following intended uses https://www.beckmancoulter.com/eCatalog/Catalog/Disease\_Management): albumin, to evaluate the hepatic/renal function, infectious diseases, inflammatory responses or nutritional assesment; alpha 1 acid glycoprotein, to assess the existence of infectious diseases or inflammation; alpha 1 antitrypsin, to check the presence of infectious diseases or inflammation; alpha 2 macroglobulin and antithrombin III, to detect anemia, thrombofilia or to assay the hepatic function; ApoA1 and ApoB, to measure the cardiovascular risk; ceruloplasmin, for the evaluation of the hepatic function or the detection of infection/inflammation; complement factors (C3 and C4), to evaluate the liver function or unmask autoimmune or inflammatory processes; fibrinogen, for thrombofilia and bledding disorders detection; haptoglobin, to uncover infectious diseases, inflammation, and anemia; plasminogen, to assess fibrinolysis; transferrin, to value the presence of anemia, the nutritional status, the presence of infectious diseases, or the renal function; and finally transthyretin/prealbumin, for hepatic function and nutritional assesment. In addition, detection of beta 2 microglobulin (e.g. Quantikine® IVD® Human 2M Immunoassay, R&D) is an aid in the diagnosis of autoimmune diseases (rheumatoid arthritis, systemic lupus erithematosus), viral infections or reduced glomerular filtration rates (kidney diseases). On the other hand, low levels of plasminogen activator inhibitor 1 (PAI-1) are linked to bleeding, while its elevation is related with an increased number of blood clots (surgery, infection, diabetes) and elevated risk of heart attack or coronary artery disease

Company name Company name 1 Abaxis, Inc ABX 40 Health Chem Diagnostics,

3 Access Bio, Inc ACC 42 Human Diagnostics

2 Abbott Laboratories ABL 41 Horiba Abx **HOR**

4 Affinity Biologicals, Inc AFF 43 Ibl, Gmbh **IBL**

6 American Diagnostica, Inc AMER 45 Immuno, Gmbh **IMM**

9 Arlington Scientific, Inc ARL 48 Kamiya Biomedical, Co **KB**

Ltd AS 49 Kent Laboratories, Inc **KL**  11 Bacton Assay Systems BAS 50 Medical Diagnostic **MDT**

7 Amico Lab, Inc AMI 46 Instrumentation Laboratory,

8 Arkray, Inc ARK 47 Jas Diagnostics Systems,

Llc **HCD**

Worldwide **HDW**

Co **ILAB**

Gmbh **JAS**

AWDT 44 Immunostics, Inc **IMMTICS**

(CAD).

5

Inc

Alfa Wassermann Diagnostic Technologies,

<sup>10</sup>Axes-Shield Diagnostics,

Table 3. Some companies developing *in vitro* diagnostics (IVD) tests for clinical use.

This table is not expected to be a comprehensive summary of all existing IVD companies. Mainly, data have been collected from different sources, like the US Food and Drug Administration Home Page (http://www.fda.gov), ZapConnect.com, or the home page of each company.

Amongst this first group of IVD tests for APPs, C-reactive protein/CRP is, by far, the most frequent offered by different companies (Table 4). That is due to a dual use: On the one hand, a low sensitivity test for inflammation or infectious/autoimmune diseases. On the other hand, systemic inflammation and CRP levels increase as the amount of visceral adipose tissue and the waist circumference become excessive, and that is because CRP (as well as the fibrinolysis inhibitor PAI-1) are not only produced by liver but also by adipocytes (Libby et al., 2010). Based on that, a high-sensitivity assay for cardiovascular risk (denoted hs-CRP) has been developed to predict future clinical cardiovascular events. Elevation of average hs-CRP level is linked to atheroesclerosis, and therefore point to a higher potential of stroke, myocardial infarction or severe peripheral vascular disease development. Thus, apparently healthy people with some inflammation (above median hsCRP) but below median levels of low-density lipoprotein (LDL) could nonetheless benefit from statin therapy (Libby et al., 2010).

Moreover, these companies are continuosly adding new products to their growing portfolio of IVD assays, as it is the case for some acute phase reactants (Table 4). For example, tests for factor VIII (hemophilia A) and von Willebrand (von Willebrand disease) are included within the pannels of reagents to check hemostasis, and alpha 1 microglobulin is been introduced in market for the functional evaluation of liver and kidney, diabetes and stroke. As well, elevation of calcitonin precursor levels in plasma represent a high risk for progression to severe sepsis if the measurement of this acute phase reactant is done shortly after the systemic infection process has started (on the first day). Still, there are other IVD tests that have not been included in Table 4, like properdin Factor B (autoimmune diseases) or apolipoprotein (a)(cardiovascular risk), that are also manufactured by some companies.

A large majority of APPs are typically measured by enzyme immunoassays (EIA, ELISA), but also by immunoturbidimetric or nephelometric technologies through chemistry analyzers such as Alfa Wassermann Alera/ACE, Roche/Hitachi, Roche/Cobas, Beckman Synchron®, Beckman/Olympus, Abbot Aeroset, Siemens/Bayer Advia or Siemens/Dade Dimension. There are some radioimmuno- (RIA) (e.g., Abbott Beta-2-Microglobulin RIA) and chromogenic assays too (e.g., BCG method for albumin), and some of these analytes have waived versions (simple and accurate, according to the CLIA complexity classification; e.g., Abaxis, Inc).

#### **4. New methods and technologies for the quantification of acute phase proteins: APPs as disease biomarkers**

The high-throughput genomic and proteomic technologies, combined with bioinformatics, give the most recent approaches to the study and analysis of APPs in human body fluids (plasma, serum). There are different methods that permit the simultaneous qualitative/quantitative analysis of several APPs from small volume of samples: 2-DE/MS, 2-DE-DIGE/MS, MALDI-TOF-MS, SELDI-TOF-MS, label-free LC-MS profiling, CE-ESI-MS, isotope tagging/MS/MS, or antibody arrays for serum profiling. Proteomic methods can now detect more than 1000 proteins in plasma at the same time. However, the comprehensive analysis of the proteome of any body fluid is still beyond our reach, despite great methodological advances in recent years. Several major challenges must be faced by researchers. For instance, proteomics of body fluids is limited to a ~g/L (ng/mL)

Amongst this first group of IVD tests for APPs, C-reactive protein/CRP is, by far, the most frequent offered by different companies (Table 4). That is due to a dual use: On the one hand, a low sensitivity test for inflammation or infectious/autoimmune diseases. On the other hand, systemic inflammation and CRP levels increase as the amount of visceral adipose tissue and the waist circumference become excessive, and that is because CRP (as well as the fibrinolysis inhibitor PAI-1) are not only produced by liver but also by adipocytes (Libby et al., 2010). Based on that, a high-sensitivity assay for cardiovascular risk (denoted hs-CRP) has been developed to predict future clinical cardiovascular events. Elevation of average hs-CRP level is linked to atheroesclerosis, and therefore point to a higher potential of stroke, myocardial infarction or severe peripheral vascular disease development. Thus, apparently healthy people with some inflammation (above median hsCRP) but below median levels of low-density lipoprotein (LDL) could nonetheless benefit

Moreover, these companies are continuosly adding new products to their growing portfolio of IVD assays, as it is the case for some acute phase reactants (Table 4). For example, tests for factor VIII (hemophilia A) and von Willebrand (von Willebrand disease) are included within the pannels of reagents to check hemostasis, and alpha 1 microglobulin is been introduced in market for the functional evaluation of liver and kidney, diabetes and stroke. As well, elevation of calcitonin precursor levels in plasma represent a high risk for progression to severe sepsis if the measurement of this acute phase reactant is done shortly after the systemic infection process has started (on the first day). Still, there are other IVD tests that have not been included in Table 4, like properdin Factor B (autoimmune diseases) or apolipoprotein (a)(cardiovascular risk), that are also manufactured by some companies. A large majority of APPs are typically measured by enzyme immunoassays (EIA, ELISA), but also by immunoturbidimetric or nephelometric technologies through chemistry analyzers such as Alfa Wassermann Alera/ACE, Roche/Hitachi, Roche/Cobas, Beckman Synchron®, Beckman/Olympus, Abbot Aeroset, Siemens/Bayer Advia or Siemens/Dade Dimension. There are some radioimmuno- (RIA) (e.g., Abbott Beta-2-Microglobulin RIA) and chromogenic assays too (e.g., BCG method for albumin), and some of these analytes have waived versions (simple and accurate, according to the CLIA complexity classification;

**4. New methods and technologies for the quantification of acute phase**

The high-throughput genomic and proteomic technologies, combined with bioinformatics, give the most recent approaches to the study and analysis of APPs in human body fluids (plasma, serum). There are different methods that permit the simultaneous qualitative/quantitative analysis of several APPs from small volume of samples: 2-DE/MS, 2-DE-DIGE/MS, MALDI-TOF-MS, SELDI-TOF-MS, label-free LC-MS profiling, CE-ESI-MS, isotope tagging/MS/MS, or antibody arrays for serum profiling. Proteomic methods can now detect more than 1000 proteins in plasma at the same time. However, the comprehensive analysis of the proteome of any body fluid is still beyond our reach, despite great methodological advances in recent years. Several major challenges must be faced by researchers.  Data come from different sources, like the US Food and Drug Administration Home Page (http://www.fda.gov), ZapConnect.com, or the home page of each company. Sometimes this one does not offer directly IVD tests for some APPs, but can do it through strategic alliances that are not reflected in Table 3 (e.g., Beckman Coulter and Instrumentation Laboratory and the HemosIL® Fibrinogen-C kit). Abbreviations used for the name of the companies are the same as in Table 3. Abbreviations for APPs are the same as in Table 1.

 *In vitro* diagnostic (IVD) tests for the quantitative determination of acute phase

This table is not intended to be a comprehensive summary. Data come from different sources, like the US Food and Drug Administration Home Page (http://www.fda.gov), ZapConnect.com, or the home page of each company. Sometimes this one does not offer directly IVD tests for some APPs, but can do it through strategic alliances that are not reflected in Table 3 (e.g., Beckman Coulter and Instrumentation Laboratory and the HemosIL® Fibrinogen-C kit). Abbreviations used for the name of the companies are the

**STG** X X

**TB** X X X X

same as in Table 3. Abbreviations for APPs are the same as in Table 1.

**VMS** X

**R2**

sensitivity, which is roughly 1000-fold less sensitive than the most specific immunoassays. Another additional problem is the range of protein concentrations in body fluids. The dynamic range of plasma proteins concentrations spams from that much as 40 mg/mL for albumin (50% of the total mass of plasma proteins) to less than 4 pg/mL; i.e., the concentration of many cytokines. Therefore, we are talking about 10-12 orders of magnitude, which means that disease-mediated alterations in soluble mediators (e.g., cytokines), or modifications in those released into the circulation through cell-leaking processes during normal cell turnover (apoptosis) or specific injuries (e.g., myocardial infarction), are hardly detectable. For example, the most common technology for fractionating and identifying proteins, 2-DE, has a range of detection of no more than 3-4 orders of magnitude with differential gel electrophoresis (DIGE). About 99% of total protein mass in plasma depends on only 22 abundant species, like albumin (35-45 mg/mL), IgG, IgA, IgM (12-18 mg/mL), 2 and -lipoproteins (LDL; 4-7 mg/mL), fibrinogen (2-6 mg/mL), 1-antitrypsin (2-5 mg/mL), 2-macroglobulin (2-4 mg/mL), transferrin (2-3 mg/mL), 1-acid-glycoprotein (1 mg/mL), hemopexin (1 mg/mL), -lipoproteins (HDL, 0.6-1.5 mg/mL), haptoglobin (0.3-2 mg/mL), prealbumin (0.3-0.4 mg/mL) or ceruloplasmin (0.3 mg/ml). Most of them are acute phase reactants; i.e., plasma resident proteins with a molecular mass larger than the kidney filtration cutoff and large half-lives, both characteristics ensuring an extended residence time and high protein concentrations (45 mg/mL-0.001 mg/mL). Thus, this high abundance makes them easily accessible to proteomic technologies, although there are still a couple of questions to consider: complexity of this family of proteins and their timedependent concentration modulation (kinetics).

Regarding complexity, the most important APPs are usually glycoproteins. This means that APPs, like immunoglobulins, are highly heterogeneous (Anderson and Anderson, 2002). The difficulty of their analysis is even enhanced by processing events, that lead to the generation of smaller proteins or peptides from larger precursors (both the plasma and cells contains numerous proteases), or the presence of genetic variants in acute phase reactants as apoE, apoH, transferrin or haptoglobin. Two-dimensional electrophoresis (2-DE) was the first used method in clinical biofluids proteomics, and still is one of the most used nowadays for different reasons. After the introduction of high resolution 2-DE in 1975 this technique was applied to the plasma proteins (Anderson & Anderson, 1977). These authors were able to resolve by 2-DE about 300 or more spots and a number of about 40 distinct plasma proteins. This initial amount has been extended to 626 identified spots, 1966 detected spots and 69 proteins in the current Swiss 2D-Page Web site (http://expasy.org/swiss-2dpage), which may represent the limit in terms of 2-DE analysis of unfractionated plasma. Therefore, it was clear very soon than 2-DE had limitations with respect to the resolution to cope with the extraordinary dynamic range of plasma proteins concentrations and the complexity issue, apart from additional problems: low and high molecular weight, hydrophobic, and very acid or basic proteins. Still, isolectric points of APPs are mostly between pH 5 and 6, and these proteins are originated mainly from liver secretion (not cell leakage; i.e., hydrophilic) and have a medium-high molecular size. In addition, there are means to reduce sample complexity and enhance the loading capacity of 2-DE (see below). Therefore, 2-DE is a technique specially suited to screen the acute phase response, and can be even essential to determine the post-translational and genetic modifications of APPs.

Nevertheless, and largely because of novel methods of serum fractionation and MS based protein identification, after the turn of the century the number of plasma proteins that could be identified increased over time. Thus, thanks to extensive fractionation serum fractionation and tandem mass spectrometry, Adkins et al. (2002) were able to identify 490 proteins. That number suffered a huge increase in 2004, to achieve an identification of 1,175 non-redundant plasma proteins, by using multidimensional chromatography and MS analysis (Anderson et al. 2004). At present, the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) has a list of 3020 plasma proteins (http://www.hupo.org/ research/hppp/)

#### **4.1 Quantitative proteomics technologies applied to the study of APPs**

However, the elaboration of a detailed list of serum/plasma proteins provides relatively limited biological information. To understand the biology of any pathological process, quantitative analysis of these proteins is essential. Perhaps, 2-DE or 2-DE fluorescence differential gel electrophoresis (DIGE), followed by MS analysis, remains as the most commonly used method (see Table 5). DIGE is a quantitative technique that enhance the dynamic range of 2-DE up to four orders of magnitude. DIGE is commercially available from GE Healthcare, and allows the direct comparison of different samples at the same time on a single 2-DE gel. This technique involves the use of up to three different samples and three cyanide dyes (Cy2, Cy3, and Cy5). These dyes have an NHS-ester reactive group designed to covalently attach to the epsilon amino group of lysine of proteins via an amide linkage. In addition, dyes label 1-2% of all the available proteins (minimal labelling, about one dye molecule per protein), without changing their pI and adding only 0.5 kDa to the protein mass. Normally, a pooled sample, comprising equal amounts of each of the initial samples within the study, is labelled with Cy2 and used as internal control. This internal standard allows normalization and is an effective way to increase accuracy and reproducibility during quantification. On the contrary, one particular disadvantage of DIGE is the equipment required for visualization and spot excision, and another major problem with these 2-DE based methodologies is that they are labor intensive and difficult to automate. In addition, as we have just commented, they are not able to effectively mine the low abundance biofluids proteome (<100 ng/ml). Therefore, the detected proteins limit primarily to high (0.1–40 mg/ml) and medium abundance (0.1–100 g/ml) species, which is enough to study the classical acute phase reactants (e.g., CRP <10 g/ml), but does not allow to find new ones. All these limitations of 2-DE based methods are driving the development of new proteomic methodologies for serum/plasma profiling that seek to reduce the workload of 2-DE (since they use automated equipment), such as MALDI-TOF-MS, SELDI-TOF-MS, label-free LC-MS profiling, CE-ESI-MS, isotope tagging/MS/MS, or antibody arrays. Although it is outside the scope of this chapter to review comprehensively these techniques, we will go through a brief summary of some of them.

Simple adsorption/washing/desorption methods are fast, easily automated and have a widespread use (Table 5). In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) samples are digested with trypsin and deposited on the MALDI plate prior peptide mass fingerprinting (PMF) or partial sequencing of peptides (MS/MS; e.g., MALDI-TOF/TOF). In surface-enhanced laser desorption/ionization (SELDI) (e.g., ProteinChip®, Ciphergen Biosystems, Palo Alto, CA) different chromatographic surfaces (hydrophobic, hydrophilic, etc) are used on a metal array to bind a subset of proteins from a sample. The unbound proteins are washed away, the bound proteins are overlaid with an organic matrix, and finally this fraction is profiled by MS analysis (e.g., MALDI-TOF-MS), generating rather simple mass spectra which can be easily analyzed. SELDI is a very useful method for high throughput screening, although this approach is not very reproducible and

fractionation and tandem mass spectrometry, Adkins et al. (2002) were able to identify 490 proteins. That number suffered a huge increase in 2004, to achieve an identification of 1,175 non-redundant plasma proteins, by using multidimensional chromatography and MS analysis (Anderson et al. 2004). At present, the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) has a list of 3020 plasma proteins (http://www.hupo.org/