Electrophoresis of the proteins in the serum is used to divide these proteins into their main fractions (albumin – a class of water-soluble proteins – and globulins, which can be subdivided into α, β, and γ types) and quantify each of the fractions. This can provide diagnostic information on disorders characterized by alterations of plasma proteins (dysproteinaemias).
At least six fractions can be recognized in the electrophoretic trace of serum from dogs and cats (Fig. 1): albumin, α1, α2, β1, β2 and γ-globulins. Each of these fractions corresponds to groups of proteins (and not single proteins) with similar physico-chemical properties. These fractions are described below.
ALBUMIN
Albumin is the most abundant class of proteins accounting for the largest electrophoretic fraction (about 50% of the plasma proteins) and has numerous functions: maintenance of oncotic or colloid-oncotic pressure, transport of molecules and drugs, buffer activity, etc.
These proteins are produced in the liver from amino acids of dietary origin or amino acids derived from the catabolism of other proteins in the body. They have a half-life of about 20 days, after which they are broken down into their component amino acids which are recovered and re-used for the synthesis of new proteins or converted into other organic compounds (for example, the gluconeogenic amino acids can be converted into glucose and used to produce energy).
These proteins can be identified by electrophoresis, but also by spectrophotometry, using the method. Biochemical identification may, however, be less accurate than the electrophoretic one and more subject to pre-analytic and analytic errors.
TOTAL GLOBULINS
The globulins constitute about 50% of proteins in the plasma and are divided into three main electrophoretic fractions α, β and γ. The amount of these proteins can be determined by electrophoresis or calculated starting from the concentrations of total proteins and albumin, measured by spectrophotometry, using the following formula:
Globulins = total proteins - albumin
α-globulins
These are almost exclusively produced by the liver and in physiological conditions have a half-life similar to that of albumin. In most species this group of globulins is subdivided into α1 (fast-migrating) and α2 (slow-migrating) forms and, using high resolution systems (on gel or with the capillary technique), further peaks can be recognized (α1-1, α1-2, etc.). The main proteins migrating as α-globulins are:
- lipoproteins: the α-lipoproteins (HDL) migrate in α1, the pre-β-lipoproteins (VLDL) migrate in α2, and the β-lipoproteins (LDL) migrate in α2 in cellulose acetate and gel (while they migrate among the β-globulins on less selective supports)
- positive acute phase proteins such as α1-antitrypsin, α1-acid glycoprotein (AGP), α2-macroglobulin, haptoglobin (Hp), caeruloplasmin (Cp) and serum amyloid A (SAA)
β-globulins
These include proteins of hepatic origin (some acute phase proteins such as complement fractions, ferritin, C-reactive protein (CRP), fibrinogen when samples of plasma rather than serum are studied) and proteins produced by the immune system, such as IgM and IgA antibodies. The most common electrophoretic techniques differentiate β1 (fast-migrating) and β2 (slow-migrating) fractions. When high resolution systems are used, other fractions (for example, β3) or subfractions, of currently unknown clinical significance in veterinary medicine, can be identified.
γ-globulins
These are almost exclusively produced by the immune system and are mainly the type IgG antibodies and some IgA, IgM and IgE. High resolution systems can distinguish γ1 (fast-migrating, containing antibodies other than IgG) and γ2 (slow-migrating, containing mostly IgG) fractions. The correct identification of antibody classes present does, however, require the use of immunoelectrophoretic techniques.
PRE-ANALYTIC ARTEFACTS
Electrophoresis can be affected by the following pre-analytic errors:
- haemolysis: haemoglobin migrates among the b-globulins and haemolysed samples can show false β peaks
- lipaemia: an increase in lipoproteins may, rarely, cause false increases in the α1 or α2-globulins
- presence of fibrinogen (in the plasma): fibrinogen migrates between the β2- and γ-globulins, creating a β-γ bridge. For this reason, electrophoresis should never be carried out on plasma. If plasma is the only sample available, much of the fibrinogen can be removed by heating the plasma to 56°C before carrying out the electrophoresis or mixing nine parts of plasma with one part of ethanol and, after 15 minutes in ice, centrifuging and using the supernatant to carry out the electrophoresis.
ALTERATIONS OF PLASMA PROTEINS (DYSPROTEINAEMIAS)
Alterations of plasma proteins can indicate quantitative changes (hyper- or hypo-proteinaemia) or qualitative changes (of one or more electrophoretic class).
Alterations of plasma proteins can also be classified as:
- "non-selective’: the ratio of albumin to globulins (A/G) remains unaltered, since there is a proportionally equal increase or decrease of all the electrophoretic fractions, leading to an increase or decrease in the concentration of total proteins.
- ‘selective’: the A/G ratio is altered because only some electrophoretic fractions increase or decrease. Although in most cases the increase or decrease of one or more of the electrophoretic classes causes hyper- or hypo-proteinaemia, respectively, it is possible that an increase in one fraction is accompanied by a decrease in another so that the concentration of total proteins could even remain within the norm. For this reason electrophoresis of serum proteins should be carried out, independently of the concentration of total proteins, in every case that the A/G is altered, or better still, in all cases in which there is a suspicion of a disease potentially capable of inducing electrophoretic alterations.
HYPERPROTEINAEMIA
The term hyperproteinaemia means an increase in the concentration of the total plasma proteins.
Non-selective hyperproteinaemia (panhyperproteinaemia)
These cases of hyperproteinemia are those in which there are equal increases in albumin and globulins and the A/G ratio remains unaltered. The main cause of a non-selective increase in proteins is dehydration or haemoconcentration. In this case the increase is relative since it is not actually the amount of proteins in the plasma that is altered, but rather their concentration because the amount of fluid in which the proteins are suspended is decreased. This condition is usually associated with a proportional increase in the haematocrit, except in anaemic subjects.
Selective hyperproteinaemia with an increase in the A/G ratio
These cases are usually due to hyperalbuminaemia, which can be a consequence of:
- dehydration or haemoconcentration, if the increase in albumin exceeds that of the globulins.
- high concentrations of corticosteroids – whether exogenous (iatrogenic administration) or endogenous (severe stress, hyperadrenocorticism) – which can induce hepatic synthesis of albumin or increase the half-life of plasma albumin.
- hepatic tumours in which the neoplastic hepatocytes increase the synthesis of albumin.
More rarely the increase in the A/G ratio depends on a decrease in globulin. It is rare to see hyperproteinaemia in these cases; indeed, hypoproteinaemia is more common (see later).
Selective hyperproteinemia with a decrease in the A/G ratio
These cases are usually due to an increase in one or more of the globulin fractions. The clinical relevance depends on which of the fractions is primarily increased. Often the increase in the globulins is associated with a decrease in albumin. If this decrease is particularly marked and the increase in the g-globulins is modest, the concentration of total proteins does not increase or even decreases (see later). Conditions characterized by selective hyperproteinaemia with an increase in globulins and a possible decrease in albumin are:
- Inflammatory processes, particularly acute ones (abscesses, pyometra, etc.), but also subacute and chronic ones and those consequent to a primarily non-inflammatory disease (for example, tumours): these particularly increase the α-globulins (Fig. 2) and/or the β-globulins (Fig. 3). Many of the positive acute phase proteins migrate in these fractions, including α2–macroglobulin and haptoglobin, which are the main proteins responsible for an increase in α2-globulins, and complement fractions (which contribute to the increase of β-globulins). Other positive acute phase proteins, although increasing notably in serum, do not contribute significantly to the α or β peaks because, in physiological conditions, they are present at very low concentrations. Furthermore, during inflammation, albumin can decrease because it escapes from vessels as a consequence of the increased vessel wall permeability that characterizes inflammatory states, and because the hepatic production of albumin (negative acute phase proteins) decreases. It should, however, be remembered that the decrease in albumin may not be particularly marked, and, therefore, the finding of hyperglobulinaemia but not of hypoalbuminaemia does not exclude the possibility that there is ongoing inflammation. If the inflammatory process has an immune-mediated component (for example, leishmaniasis, ehrlichiosis, infective peritonitis), not only may the α-globulins (and possibly the β globulins) increase, but also the γ-globulins, causing a polyclonal gammopathy (Fig. 4) or oligoclonal gammopathy.
- Neoplasms of plasma cells (multiple myeloma, more rarely plasmacytoma) or lymphoid cells (lymphoma). The clonal proliferation of these cells leads to an overproduction of a single type of antibody which causes monoclonal gammopathy (Fig. 5).
Fig. 2 Fig. 3 Fig. 4 Fig. 5
HYPOPROTEINAEMIA
Hypoproteinaemia means a decrease in the concentration of total plasma proteins.
Non-selective hypoproteinaemia (panhypoproteinaemia)
These are cases in which both the albumin and globulins decrease equally and the A/G ratio remains unchanged. The main cause of non-selective hypoproteinaemia is a protein-losing enteropathy, in which dietary proteins are not absorbed or even already formed blood proteins of all electrophoretic fractions are lost in the intestinal lumen. This situation occurs in idiopathic conditions (for example, lymphangiectasia), in some inflammatory disorders (for example, inflammatory bowel disease) and, to a lesser degree, in cases of prolonged fasting, malabsorption or maldigestion, in which the diminished absorption prevails over the loss of proteins in the intestinal lumen. It is, however, possible that in protein-losing enteropathies of an inflammatory nature, the loss of proteins coexists with production of proteins in response to the inflammatory stimulus: consequently, hypoalbuminaemia may be very marked, while hypoglobulinaemia may be mild or absent: the hypoproteinaemia can, therefore, be selective (see later) or the concentration of total proteins may remain within the norm or, in rare cases, actually increase slightly.
Another possible cause of non-selective hypoproteinaemia is severe liver disease, characterized by hepatic failure (chronic liver diseases, porto-systemic shunts, microvascular dysplasia). In these conditions the main problem is a decrease in the synthesis of albumin, such that the most common problem is selective hypoproteinaemia (see later). If, however, the liver disease is particularly severe, the hepatic production of α- and β-globulins may also be reduced and a non-selective hypoproteinaemia may, therefore, be observed.
Selective hypoproteinaemia with an increase in the A/G ratio
In these cases the problem is a predominant decrease in globulins. These very rare conditions are usually caused by a decrease in the globulins produced by the immune system. This occurs in congenital immunodeficiency disorders (for example, inherited agammaglobulinaemia) or, more frequently, in acquired conditions (for example, administration of immunosuppressive doses of corticosteroids or immunodepressive drugs). It is a physiological condition in neonates and resolves spontaneously with the intake of colostrum. However, in the absence of colostrum it can become persistent and pathological.
Selective hypoproteinaemia with a decrease in the A/G ratio
These are more frequent forms of hypoproteinaemia, in which there is mainly a decrease in albumin or, at any rate, albumin decreases more than globulins. The most common causes are described below.
- Intestinal disorders: while in the true protein-losing enteropathies there are decreases in both albumin and globulins, in less severe forms of gastrointestinal disease or in disorders with an inflammatory component, globulin levels can remain within the normal range and, if the loss of albumin is very marked, hypoproteinaemia, characterized by severe hypoalbuminaemia can be observed.
- Chronic liver diseases/liver failure: except in the most serious forms in which the production of all classes of proteins in lost, during liver failure there is a notable selective decrease in the synthesis of albumin, which leads to severe hypoalbuminaemia. In order to avoid the decrease in oncotic pressure resulting from the hypoalbuminaemia, the extra-hepatic production of proteins (β- and γ-globulins, produced by the immune system) is increased. This production is favoured by the fact that the lack of detoxifying activity of the liver leads to an accumulation of toxic metabolites that provoke an immune and/or antibody response. These mechanisms underlie the appearance of the so-called β- γ bridge that can be observed in cases of liver cirrhosis.
- Glomerulopathies or protein-losing nephropathies: in cases of renal damage (glomerulonephritis, amyloidosis), the permeability of the glomerulus increases and there is a predominant loss of albumin, leading to severe hypoalbuminaemia. If the glomerular damage is more severe, higher molecular weight proteins, belonging to the globulin fractions, are also lost. Some globulins, however, are very bulky (for example, α2–macroglobulin and some β-globulins) and remain in the circulation even in the case of severe glomerular damage: consequently the percentages of α2- and β2-globulins may be increased causing the typical picture of nephrotic syndrome that accompanies protein-losing nephropathies (hypoproteinaemia, hypoalbuminaemia, increased percentages of β2 and/or α2-globulins, selective and non-selective glomerular proteinuria);
- Inflammation: although inflammation more usually causes non-selective hyperproteinaemia (see above) in cases of particularly severe hypoalbuminaemia, a selective hypoproteinaemia with a decrease in the A/G ratio may rarely be observed.
GAMMOPATHIES
The electrophoretic changes caused by increases in the concentration of immunoglobulins are called “gammopathies”. If the IgG increase, which migrate in the γ fraction, there is a real gammopathy, that is, an increase in the height and/or width of the peak of the γ-globulins. If, on the other hand, the increase is in IgM or IgA, which migrate in the β fraction, there is an increase in the height and/or width of the peak of β-globulins, also called “gammopathy” even though the γ fraction is not involved. Gammopathies may have a reactive origin (polyclonal gammopathies) or a neoplastic cause (monoclonal gammopathies).
Polyclonal gammopathies
These occur in response to immune stimuli such as infectious agents or auto-antigens. In these cases there is rarely a single antigen involved but rather several epitopes capable of provoking antibody responses, particularly in the case of infectious agents. Thus, various antibodies with different physicochemical characteristics are produced. The result is a particularly broad peak (wider than that of albumin) whose height varies depending on the concentration of the antibodies. This peak is found in the β fraction (initial stages of the antibody response, in which IgM are produced) or, more frequently, in the γ fraction (if due to IgG). A similar situation can also occur in elderly animals or animals living in overcrowded conditions, in which there is recirculation of microbial agents that cause chronic stimulation of the immune system, or following vaccination. In spontaneous forms (for example, feline infectious peritonitis, leishmaniasis), polyclonal gammopathies are often associated with increases in other electrophoretic fractions indicative of ongoing inflammation (for example, α- or β-globulins).
Monoclonal gammopathies
When a single clone of lymphocytes or plasma cells replicates uncontrollably, very similar antibodies, which have the same physicochemical characteristics, are produced. Given that it is these characteristics that determine electrophoretic migration, the resulting peak, defined as monoclonal, is very high and its width is similar to or less than that of albumin. This peak is in the γ fraction if it consists of IgG and in the β fraction if it consists of IgM or IgA. Monoclonal gammopathies usually occur in subjects with plasma cell tumours (multiple myeloma, plasmacytoma, Waldenström’s macroglobulinaemia) or B-lymphocyte neoplasms (lymphomas, lymphocytic leukaemias). In human medicine there is also an idiopathic form not associated with a definitive tumour, called monoclonal gammopathy of unknown significance (MGUS).
Biclonal or oligoclonal gammopathies
Sometimes the infectious agents that stimulate the immune response are formed of only a few antigens. The result is an oligoclonal peak, whose width is intermediate between that of polyclonal and monoclonal peaks, although often difficult to differentiate from monoclonal peaks, particularly if evaluated by capillary electrophoresis, which tends to “squeeze” and elongate the electrophoretic peaks. Likewise, during a malignancy a small number of lymphocytic clones, instead of a single clone, may develop. In this case, two or three monoclonal-like peaks (bi- or tri-clonal gammopathy, respectively) are formed: these are clearly visible by capillary electrophoresis, although less easily differentiated in gel or acetate electrophoresis where they often appear as oligoclonal.
The analytical principle of capillary electrophoresis differs from that of traditional zonal electrophoresis: instead of being placed on a support, the serum is diluted in a buffer. The proteins therefore remain in a liquid phase and are placed in a long silica capillary in which an electroendosmotic flow, which is stronger than the electrical field, drags the proteins towards the cathode (in the direction opposite to that in traditional electrophoresis), until they reach the spectrophotometric detection system. The proteins passing through this detector are recognized on the basis of their wave-length. The instrument then generates an electrophoretic trace similar to that obtained with traditional techniques, in which the width of the peaks corresponds to the duration of the signal received and their height corresponds to the intensity of the signal.
The greater resolution of this method does, however, often produce small morphological differences compared to the traces obtained with the traditional techniques (Fig. 6). In particular, it is often possible to detect more fractions than the six normally detectable in traditional electrophoresis. Furthermore, the peaks referable to the α- and β-globulins often have a very jagged appearance, due to the presence of “minor” peaks caused by proteins that have not yet been identified in veterinary medicine. Finally, the peaks obtained with capillary electrophoresis often seem narrower and higher than those obtained with traditional electrophoresis. It is, therefore, essential to know which technique was used to produce the trace in order to be able to interpret it correctly.