**4. Physiologic serum protein pattern in large and small ruminants**

Following electrophoresis, serum proteins can be separated into four basic fractions including albumin, alpha(α)-, beta(β)-, and gamma(γ)-globulins [100]. Each band consisted of many individual proteins having various metabolic activities. The electrophoretic pattern of serum proteins and its interpretation are related to differences observed among various animal species, as well as among different groups of animals. Great species-specific variations in the type and size of serum protein fractions were observed by many researchers [101, 102].The number, shape, and size of fractions and subfractions change a lot with the animal species and breed [103]: the most important differences are inside β-globulins and even γ-globulins. Differences in the electrophoretic mobility of serum proteins have been observed also between ruminant species (**Figure 2**).

Transthyretin is a small globular non-glycosylated tryptophan-rich protein of a homotetrameric structure, composed of four identical subunits with two thyroxine binding sites per tetramer [111]. The main physiological functions of TTR include the carriage of thyroid hormones [112]. Another important function of TTR is the transport of retinol (vitamin A) through its association with retinol-binding protein (RBP) from its main storage site in the liver to target cells [113]. From this reason, in the 1980s, the name prealbumin was changed to transthyretin (TTR) describing its ability to bind both thyroid hormones, and retinol binding protein (RBP) [114]. Furthermore, transthyretin acts as a negative acute-phase reactant, serum concentrations of which fall due to decreased synthesis in inflammation, trauma, tissue injury, or stress [115]. It is synthesized mainly by hepatic parenchymal cells and in the choroid plexus of the brain, which has the highest concentration of TTR in the body [116, 117]. In cerebrospinal fluid, it is the second most abundant protein [118]. The major sites of transthyretin degradation are the liver, muscles, and skin [119]. It has a half-life in blood serum of approximately 2 days, which is much shorter than that of albumin [120]. Transthyretin is, therefore, more sensitive to changes in protein-energy status, and thus may be used as an indicator of

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The concentrations of TTR in blood serum may be affected by many factors, including age, gender, as well as blood-drawing methods. A marked increase of TTR values from 72.9 to 251.4 mg/l was observed by Tóthová et al. [122] in calves 1 day after colostrum intake with a consecutive gradual decrease till the end of the third month of life. Rona [123] described that bovine colostrum contains, among other bioactive molecules, a small amount of prealbumin (transthyretin). Thus, the increase of serum TTR concentrations observed in calves after colostrum intake may reflect the adequate nutrition, as well as its hepatic synthesis due to adequate protein and energy intake [112]. The effect of hormonal changes during pregnancy on the concentrations of TTR in animals has not been reported. Our findings suggest no significant changes in TTR concentrations during the last week of pregnancy and early stages of lactation in dairy cows (unpublished data). The usefulness of prealbumin in the clinical and laboratory diagnosis of diseases was evaluated in dogs with nonthyroidal illness (including neoplasia, allergy, cardiac disease, gastrointestinal disease, parasitism, and hepatic disease) and in pigs with *Streptococcus suis* type 2 infection, showing its lower concentrations compared with healthy ones [124, 125]. In cattle, there are very little published reports about the use of prealbumin in the diagnosis of diseases. Our preliminary results suggest lower concentrations of TTR in diarrheic calves at the age of 1 month compared with healthy animals at the same age. Similarly, *Mycobacterium avium paratuberculosis* seropositive cows showed lower

Albumin is the most abundant protein found in blood plasma or serum, and essential part of the biochemistry profile. It is a homogenous protein fraction and is visible as a discrete zone on the electrophoretogram. In animals, 35–50% of the total serum protein concentration is made up from albumin [22]. The shape and size of albumin fraction are very similar in all ruminant species, which are related to its high serum concentration, homogenous electric charge, and high staining affinity. However, there are great differences in its relative

TTR values than those obtained in healthy cattle (unpublished data).

malnutrition [121].

**4.2. Albumin**

Nagy et al. [104], by using agarose gel electrophoresis, described six fractions in bovine serum comprising albumin, α<sup>1</sup> - and α<sup>2</sup> -, β<sup>1</sup> - and β<sup>2</sup> -, and γ-globulins. Whereas, Alberghina et al. [105] and Piccione et al. [106] separated the bovine serum proteins into five fractions, comprising albumin, α<sup>1</sup> -, α<sup>2</sup> -, β-, and γ-globulins. The number of protein fractions in sheep and goat serum varied between various authors. Nagy et al. [104] and Esmaeilnejad et al. [74] in sheep serum recorded albumin, α<sup>1</sup> -, α<sup>2</sup> -, β-, γ<sup>1</sup> -, and γ<sup>2</sup> -globulins, while the goat serum proteins showed albumin, α<sup>1</sup> -, α<sup>2</sup> -, β-, and γ-globulin fractions [104, 107]. In contrast, Cyrillo et al. [108], Fernandez et al. [109], and Alberghina et al. [102] determined only one α-globulin and two β-globulin fractions in goat serum.

#### **4.1. Prealbumin (transthyretin)**

Prealbumin (transthyretin, TTR) is the most rapidly migrating protein fraction in serum visible as a band anodic to the main albumin fraction on the electrophoretic gels [79]. According to Hamilton and Benson [110], this property is attributed to human prealbumin, not to bovine. Kaneko [22] stated also that prealbumin is not always visualized in electrophoretograms and may not exist in all animal species, including ruminant species. Therefore, in these animals, species-specific ELISA assays should be used for the detection and quantification of transthyretin.

**Figure 2.** Representative agar gel electrophoretogram in a cow (a), sheep (b), and goat (c) [104].

Transthyretin is a small globular non-glycosylated tryptophan-rich protein of a homotetrameric structure, composed of four identical subunits with two thyroxine binding sites per tetramer [111]. The main physiological functions of TTR include the carriage of thyroid hormones [112]. Another important function of TTR is the transport of retinol (vitamin A) through its association with retinol-binding protein (RBP) from its main storage site in the liver to target cells [113]. From this reason, in the 1980s, the name prealbumin was changed to transthyretin (TTR) describing its ability to bind both thyroid hormones, and retinol binding protein (RBP) [114]. Furthermore, transthyretin acts as a negative acute-phase reactant, serum concentrations of which fall due to decreased synthesis in inflammation, trauma, tissue injury, or stress [115]. It is synthesized mainly by hepatic parenchymal cells and in the choroid plexus of the brain, which has the highest concentration of TTR in the body [116, 117]. In cerebrospinal fluid, it is the second most abundant protein [118]. The major sites of transthyretin degradation are the liver, muscles, and skin [119]. It has a half-life in blood serum of approximately 2 days, which is much shorter than that of albumin [120]. Transthyretin is, therefore, more sensitive to changes in protein-energy status, and thus may be used as an indicator of malnutrition [121].

The concentrations of TTR in blood serum may be affected by many factors, including age, gender, as well as blood-drawing methods. A marked increase of TTR values from 72.9 to 251.4 mg/l was observed by Tóthová et al. [122] in calves 1 day after colostrum intake with a consecutive gradual decrease till the end of the third month of life. Rona [123] described that bovine colostrum contains, among other bioactive molecules, a small amount of prealbumin (transthyretin). Thus, the increase of serum TTR concentrations observed in calves after colostrum intake may reflect the adequate nutrition, as well as its hepatic synthesis due to adequate protein and energy intake [112]. The effect of hormonal changes during pregnancy on the concentrations of TTR in animals has not been reported. Our findings suggest no significant changes in TTR concentrations during the last week of pregnancy and early stages of lactation in dairy cows (unpublished data). The usefulness of prealbumin in the clinical and laboratory diagnosis of diseases was evaluated in dogs with nonthyroidal illness (including neoplasia, allergy, cardiac disease, gastrointestinal disease, parasitism, and hepatic disease) and in pigs with *Streptococcus suis* type 2 infection, showing its lower concentrations compared with healthy ones [124, 125]. In cattle, there are very little published reports about the use of prealbumin in the diagnosis of diseases. Our preliminary results suggest lower concentrations of TTR in diarrheic calves at the age of 1 month compared with healthy animals at the same age. Similarly, *Mycobacterium avium paratuberculosis* seropositive cows showed lower TTR values than those obtained in healthy cattle (unpublished data).

#### **4.2. Albumin**

**4. Physiologic serum protein pattern in large and small ruminants**

ruminant species (**Figure 2**).


112 Ruminants - The Husbandry, Economic and Health Aspects


two β-globulin fractions in goat serum.

serum recorded albumin, α<sup>1</sup>

**4.1. Prealbumin (transthyretin)**






**Figure 2.** Representative agar gel electrophoretogram in a cow (a), sheep (b), and goat (c) [104].

comprising albumin, α<sup>1</sup>

ing albumin, α<sup>1</sup>

transthyretin.

showed albumin, α<sup>1</sup>

Following electrophoresis, serum proteins can be separated into four basic fractions including albumin, alpha(α)-, beta(β)-, and gamma(γ)-globulins [100]. Each band consisted of many individual proteins having various metabolic activities. The electrophoretic pattern of serum proteins and its interpretation are related to differences observed among various animal species, as well as among different groups of animals. Great species-specific variations in the type and size of serum protein fractions were observed by many researchers [101, 102].The number, shape, and size of fractions and subfractions change a lot with the animal species and breed [103]: the most important differences are inside β-globulins and even γ-globulins. Differences in the electrophoretic mobility of serum proteins have been observed also between

Nagy et al. [104], by using agarose gel electrophoresis, described six fractions in bovine serum

and Piccione et al. [106] separated the bovine serum proteins into five fractions, compris-

serum varied between various authors. Nagy et al. [104] and Esmaeilnejad et al. [74] in sheep

[108], Fernandez et al. [109], and Alberghina et al. [102] determined only one α-globulin and

Prealbumin (transthyretin, TTR) is the most rapidly migrating protein fraction in serum visible as a band anodic to the main albumin fraction on the electrophoretic gels [79]. According to Hamilton and Benson [110], this property is attributed to human prealbumin, not to bovine. Kaneko [22] stated also that prealbumin is not always visualized in electrophoretograms and may not exist in all animal species, including ruminant species. Therefore, in these animals, species-specific ELISA assays should be used for the detection and quantification of






Albumin is the most abundant protein found in blood plasma or serum, and essential part of the biochemistry profile. It is a homogenous protein fraction and is visible as a discrete zone on the electrophoretogram. In animals, 35–50% of the total serum protein concentration is made up from albumin [22]. The shape and size of albumin fraction are very similar in all ruminant species, which are related to its high serum concentration, homogenous electric charge, and high staining affinity. However, there are great differences in its relative concentrations between different animal species [126]. Albumin can be seen on the left side of the electrophoretogram closest to the anode, where forms a large peak [76].

*4.3.1.1. Alpha-1 antitrypsin*

the diagnostic utility of alpha-1 antitrypsin.

identification of feline infectious peritonitis [144].

phase response by external stimuli.

*4.3.1.2. Alpha-1 acid glycoprotein*

Alpha-1 antitrypsin (AAT) is the major inhibitor of serine proteases (serpin) such as neutrophil elastase and proteinase-3 in the blood [137]. It is also an acute-phase protein. In some acute-phase inflammatory reactions, the concentrations of AAT may increase in order to limit the damage caused by activated neutrophil granulocytes and their enzyme elastase, thus limiting the tissue injury caused by proteases at the site of inflammation [138]. The clinical importance of AAT is underlined in patient with AAT deficiency, a hereditary disorder that can lead to severe tissue breakdown during inflammation [139]. Consequently, pulmonary emphysema, chronic obstructive lung disease, liver diseases, as well as liver cirrhosis may occur in these patients. In addition, liver cells produce an abnormal protein, which may accumulate in the body, leading to inflammation and/or cirrhosis of the liver [140]. From animal species, Sevelius et al. [141] measured the concentrations of alpha-1 antitrypsin in dogs and evaluated whether AAT aggregates could initiate liver disease. In cattle, little is known about

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Alpha-1 acid glycoprotein (AGP) or orosomucoid is a highly glycosylated protein of which about 45% is carbohydrate and the composition of the glycan residues is known to alter during an acute-phase response [142]. AGP is considered as a natural anti-inflammatory and immunomodulatory agent. It has also been suggested that AGP is required to maintain capillary permeability [142]. Furthermore, AGP is one of the most important drug-binding proteins in plasma that can have important pharmacokinetic implications [143]. It has a moderate acute-phase response in most animal species and is more likely associated with chronic conditions. The serum concentration of AGP may be a valuable differential diagnostic analyte in the

In ruminant species, the concentrations of AGP were evaluated by Tóthová et al. [145] in calves during the first month of life. In this study, the AGP values were roughly uniform shortly after birth with an increase of values from the day 2 of life till the end of the first month of age, probably related to the normal process of growth, exposure of animals to changing environmental conditions, and nutritional factors. Similar findings were demonstrated by Rocha et al. [146]. On the other hand, the highest concentrations of AGP in the plasma were found by Itoh et al. [147] in calves immediately after birth (1368 μg/ml), gradually decreasing to 249 ± 100 μg/ml during the first 3 days of life, which are comparable to physiological values in adult bovine. Similarly, high plasma concentrations of AGP were observed by Orro et al. [148] in calves after birth, which was followed by a decrease during the first 3 weeks of life to adult values. The very high concentrations of AGP in the fetal stages may be related to synthesis of AGP in the embryonic liver [147]. These studies indicate that the production of AGP in the neonatal period is fetally regulated and its high serum concentrations after birth are not necessarily a sign of the activation of the acute-

Albumin is small size protein with a molecular weight of 69 kDa. The main functions of albumin are the maintenance of homeostasis and transportation of substances, and it also acts as a free-radical scavenger [127]. It is responsible for about 75% of the osmotic pressure of plasma and is a major source of amino acids that can be utilized by the animal's body when necessary [128]. It also serves as a carrier protein for many insoluble organic substances (e.g., unconjugated bilirubin). Serum albumin is the major negative acute-phase protein. The synthesis of positive acute-phase proteins is markedly increased during the acute inflammatory processes. These reactions require a great amount of amino acids. Thus, albumin synthesis is downregulated and amino acids are used mainly for the synthesis of the positive acute-phase proteins [129]. Catabolism of albumin occurs in various tissues, where it enters cells by pinocytosis and is then degraded by proteases [130]. The major sites of these catabolic processes are muscle, liver, and kidney. There are major species-specific differences in the turnover of albumin, reflecting the body size. The half-time for clearance of albumin varies from 1.9 days in the mouse to 14–16 days in ruminants, and because of this, it may serve as a marker of chronic nutritional status [131]. Furthermore, may studies have established albumin as an indicator of morbidity and mortality [132].

#### **4.3. Globulins**

The globulin fractions may be found on the right side of the electrophoretogram. These peaks include a very heterogenous group of proteins, and depending on the species, there may normally be one or two α, one or two β, and one or two γ fractions [22].
