Acute phase proteins

The term acute phase proteins (APP) refers toproteins the blood concentration of which is altered by at least 25% in the course of an inflammatory process. This quantitative variation can be in the form of an increase (positive acute phase proteins) or a decrease (negative acute phase proteins).

PATHOPHYSIOLOGY

The main site of production of acute phase proteins is the liver, although it has been demonstrated that for some of them there is "secondary" production sites (e.g.: serum amyloid A in milk is also produced by cells of the mammary gland, and the one detectable in the synovial fluid corresponds to an isoform produced in the intra-articular space which differs from the haematic one).

The stimulus leading to modulation of the protein response in the course of inflammation is part of the so-called “acute phase reaction”, i.e. the series of events that the body generates when in the presence of a potentially pathogenic event.

The acute phase reaction (Fig. 1)  is caused by the release of cytokines by the phagocytes which have been recruited to the inflammation site. Neutrophils and macrophages release proinflammatory cytokines in the blood, such as interleukin 1 (IL-1), interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α). These cytokines cause a variety of systemic responses, which include leukocytosis, fever and, specifically,  the modulation of the hepatic protein synthesis. This latter phenomenon is favoured by a "permissive" effect exerted by glucocorticoids, which are also produced by stimulation of the hypothalamus-pituitary-adrenal axis by pro-inflammatory cytokines. At hepatic level, therefore, there is an increase in the synthesis of proteins used in defensive processes (positive APPs), and the inhibition of the synthesis of proteins considered either non-essential, or the circulation of which "should" in any case be reduced in order to increase the efficiency of certain metabolic systems (negative APPs).

  Fig. 1

PRINCIPAL CHEMICAL AND BIOLOGICAL FEATURES OF APPs

In light of the production kinetics described above, it follows that the main characteristic of acute phase proteins is their association with inflammatory events. However, this association is entirely non-specific: both the increase of positive APPs and the decrease of the negative ones simply indicate the presence of an "acute phase reaction", i.e. the body's response to potentially damaging stimuli. It does not matter if the events are primarily inflammatory and/or infectious  (viruses, bacteria, fungi, protozoa, immune-mediated diseases), or non-specific responses against primary non-inflammatory disorders (e.g. trauma, necrosis, tumours).

Another important feature is the variability in the intensity of the response of the different APPs: although numerous molecules exist whose blood concentration is altered significantly in the course of an inflammatory process, only some molecules increase or decrease in such a way that the increase or decrease in their blood concentration can be used for diagnostic purposes. In principle, three groups of positive APPs can be recognized, based on the amount of their increase in the blood. These groups are: 1) proteins that increase significantly but without doubling their blood concentration (e.g. fibrinogen, complement factors); 2) proteins whose blood concentration increases by 2-3 times against the baseline value (e.g. haptoglobin, caeruloplasmin); 3) proteins whose blood concentration may increase from 10 to 1000 times against the basal concentration (e.g.: C-reactive protein and serum amyloid A).

The response of positive APPs is also characterized by a certain degree of species-specificity: in every animal species it is possible to recognize one or more "major" positive APPs and other "moderate" or "minor" positive APPs. The term "major APP" refers to proteins that in a given animal species increase more frequently or to a greater extent than in others, while the  moderate or minor positive APPs are the ones that rarely increase in value, or they do so to such a minor extent that they are of limited clinical significance. The use of APPs as a diagnostic marker should therefore be based primarily on the use of the "major" proteins of the species being examined. The major and minor positive APPs of animal species of veterinary interest are reported in Table 1.

Table 1.CRP = C-reactive protein, SAA = serum amyloid A, AGP = alpha -1-acid glycoprotein; Hp = haptoglobin, Pig-MAP = Pig Major acute phase protein,  Cp =caeruloplasmin; Fib = fibrinogen, Fer = ferritin , C3 and C4 = the complement fractions C3 and C4.

In addition, depending on the kinetics of response, APPs are also defined as “fast” (characterized by an immediate increase after the stimulus, as for example in the case of  SAA or CRP) or “slow” (with the increase requiring a few hours or days, as in the case of Hp or fibrinogen among the positive APPs and  albumin among the negative APPs). (Fig. 2).

Finally, the different APPs, though characterized by different physical-chemical properties and  presenting different blood concentrations (some, like fibrinogen, are usually detectable in the blood, while others, such as SAA, are virtually absent, except in the course of an inflammatory event), share the characteristic of migrating in  electrophoresis in fractions a or b, with rare exceptions. The increase in APPs may be suspected when the serum protein electrophoresis [5] reveals the presence of alterations in the above mentioned fractions a or b, compatible with the presence of an inflammatory event.

DIAGNOSTIC UTILITY OF APP ALTERATIONS

As previously mentioned, the increase of positive APPs or the decrease of negative APPs is a nonspecific indication of the activation of the acute phase reaction and not the confirmation of the presence of a given pathology. Said differently, an elevation in positive APPs and/or a reduction in negative APPs is no evidence of the presence of a disease, but simply evidence that the body is reacting against a potential pathogen. From a diagnostic point of view this could seem like a limitation, however APPs  may be useful in case of vague and nonspecific symptoms, allowing to identify inflammatory events which are not clinically evident, or they may be used as an early marker, as the variation takes place before the appearance of clear symptoms. Moreover, given that APP variations are dependent on the release of cytokines in the site of phlogosis, their plasma levels remain elevated as long as the inflammatory stimulus continues to be present locally, even if the patient may appear to improve after supportive therapies. This indicates that the apparent clinical improvement does not correspond to an actual recovery and that, in fact, the aetiological treatment is not effective. On the contrary, APP levels will decrease with the ceasing of the phagocytic activity, allowing the early detection of animals that respond to treatment, even in the presence of lesions resulting from phlogosis which may seem to be slowing the clinical recovery. The prognostic utility of APPs has been demonstrated in particular in the dog, both in the course of  primarily inflammatory forms (e.g.  leishmaniasis, pyometra) and in the presence of tumours with secondary phlogosis.

Like any other diagnostic parameter, however, any variables that may influence the plasma concentration of the APPs must be taken into account. These include certain pathophysiological states (pregnancy and, in farm animals, lactation) or pharmacological treatments (anaesthetics, antibiotics, anticonvulsants, glucocorticoids) that may lead to changes in the plasma concentration of the APPs, even in the absence of inflammation.

PRINCIPAL POSITIVE ACUTE PHASE PROTEINS

Serum amyloid A (SAA): serum amyloid A is a positive acute phase protein involved in the protection of inflamed tissues. It works by removing the oxidized lipids (which in turn can act as oxidizing agents) that may be released in the course of tissue lesions; it also has an immunomodulatory and opsonizing action against some pathogens.

In almost all animal species serum amyloid A acts as a major positive APP. Its increase is often extremely relevant (up to 1000 times the baseline value, with the increase detectable within a few hours after the stimulus and with a peak within 24-48 hours), however among the different APPs this is the one less specifically linked to inflammatory processes, as it also increases in the course of cancer, renal failure, diabetes and other non-inflammatory forms, especially in cats.

It is commonly measured with immunological methods (e.g. ELISA, immunoturbidimetry), based on the use of reactive antibodies against preserved portions of the SAA molecule (and therefore usable in many if not in all animal species) or based on the use of species-specific antibodies.

Haptoglobin (Hp): haptoglobin is a positive acute phase protein, the primary function of which is to bind free haemoglobin in order to prevent its elimination via the kidneys and facilitate the hepatic uptake. In vitro tests have also shown the presence of some immunomodulatory properties.

Haptoglobin is the major positive APP in farm animals and its assay can be useful also in dogs. The elevation of  Hp, however, is never very high, with values that only rarely exceed 2-3 times the baseline value. Being a binding protein for haemoglobin, the Hp value may decrease in the course of haemolytic anaemia. Its hepatic synthesis may, however, increase due to induction by endogenous (e.g.: hyperadrenocorticism) or exogenous cortecosteroids.

The traditional method by which Hp is measured is with a manual or automated colorimetric assay running on standard spectrophotometers; the test relies on the bond between Hp and the haemoglobin contained in the assay. Special immunological assays are available for dogs, based on antibodies which are species-specific or cross-reactive with canine Hp.

C-reactive protein (CRP): C-reactive protein is a positive acute phase protein, the primary function of which is to bind to certain bacterial molecules in order to act as an opsonin and facilitate phagocytosis. It can however also participate in the immune response, modulating the synthesis of certain cytokines.

CRP serves as a major APP in dogs; its increase, caused by a phlogistic stimuli,  takes place in a few hours, reaching values as much as 100 times the normal value; its role in other species is barely significant.

CRP can be measured by immunoturbidimetry, using commercial assays containing antibodies for the human molecule which are cross-reactive with the canine molecule.

Alpha 1-acid glycoprotein (AGP): AGP is a positive acute phase protein having an immunomodulatory function which is probably due to its glucidic component. In fact in humans and in animals, these changes in the glucidic component are associated with forms of resistance or susceptibility to infectious disease, especially of viral origin. AGP also acts as a carrier molecule for many drugs.

Alpha 1-acid glycoprotein is the major acute phase protein in cats; its increase is moderate (3-4 times the normal values) in the course of nonspecific inflammatory processes, but it can reach concentrations higher than 10 times the baseline value in the course of feline infectious peritonitis (FIP). In dogs and in other animal species AGP behaves like a moderate-minor APP.

AGP is measured by immunological assays (ELISA, immunoturbidimetry, radial immunodiffusion) involving the use of species-specific antibodies that are not cross-reactive with the AGP of species other than the one in question.

Ceruloplasmin (Cp): ceruloplasmin acts as a positive acute phase protein, however its main function is to bind copper, preventing its loss and, above all, subtracting it from the availability of bacteria which could use it for their own metabolism. Cp also seems to possess certain immunomodulatory functions. Regardless of the species, in the course of phlogosis there is a moderate increase of ceruplasmin.

Cp can be measured with colorimetric methods adaptable to normal spectrophotometers and based on the affinity of Cp for copper, or with immunological methods based on the use of antibodies. On the whole, the immunological assays are species-specific, however some validation studies have shown a certain degree of interspecies cross-reactivity.

Ferritin: ferritin is a positive acute phase protein which plays, for iron, the same role that Cp plays for copper. It binds itself to the iron in order to prevent its loss while decreasing the availability of iron for the metabolism of any bacteria present.

Ferritin increases during inflammatory events, however it also shows significant increases in the presence of tumours. Its utility as an acute phase protein, demonstrated in humans, and to a lesser extent in dogs, is limited by the fact that its concentration tends to be proportional to the reserves of iron. Any increase in the course of inflammation may therefore be masked by its tendency to decrease in the presence of a reduced availability of iron. While this limits its use as a positive APP, the measurement of ferritin levels may still be useful for evaluating iron reserves; the ferritin assay is therefore included in the iron profile for the diagnostic/prognostic evaluation of iron-deficiency anaemia.

Ferritin is measured mainly with immunoturbidimetry, using assays containing antibodies against the human molecule, some of which are able to cross-react with canine ferritin.

Fibrinogen: besides being the final component of the coagulation cascade, fibrinogen is a mediator of phlogosis, and hence its hepatic synthesis increases in the course of phlogosis. Fibrinogen is therefore a positive acute phase protein and, although it behaves like a moderate APP in nearly all species, its role as a marker of inflammation is well recognized, particularly in horses, in which it increases a few hours after the phlogistic stimulus reaching its peak, which is usually about 2 times the normal value, after about 24 hours. A possible limitation in the use of fibrinogen as a positive APP is that the synthesis of fibrinogen tends to decrease in the course of moderate to severe liver failure. While this makes hypofibrinogenaemia (hypoinosis) a good indirect indicator of liver failure, it may also complicate the picture of inflammation, as a potential hyperfibrinogenaemia (hyperinosis) induced by phlogosis may be masked by a decreased hepatic production in cases of concomitant liver failure, or increased peripheral consumption in the course of coagulopathies (e.g. disseminated intravascular coagulation)

Precisely because of its dual role of inflammatory protein and component of the coagulation cascade, the blood concentration of fibrinogen is usually determined indirectly, measuring its activity with a coagulometer, and it is commonly included in the coagulation profile.

Pig Major Acute Phase Protein (Pig-MAP): Pig-MAP is a positive acute phase protein which acts as an antiprotease substance that protects the tissues from the activities of prosthetic enzymes released into the tissues themselves in the course of phlogosis by pathogens or by degranulation of the leukocytes.

As the name suggests, Pig-MAP plays an important role as a positive APP in pigs, a species in which it undergoes moderate to heavy increases in the course of various types of inflammatory events. It is assayed with species-specific antibodies, usually with solid phase methods (ELISA).

PRINCIPAL NEGATIVE ACUTE PHASE PROTEINS

Albumin: the role of albumin as a negative acute phase protein is amply demonstrated in humans; although not in all animal species is there experimental evidence demonstrating a reduction in the hepatic production of albumin, the decrease in the blood concentration of albumin (hypoalbuminaemia) in the course of inflammation has been reported in nearly all animal species. This suggests that even in species other than humans albumin can act as a negative APP. Indeed, given that albumin is the protein mainly present in the plasma, a reduction in its hepatic synthesis allows the body to have a greater amount of amino acids to use for the synthesis of positive APPs. As a negative APP, however, albumin is relatively "slow" because, in order to appreciate its decrease in the blood, it is first necessary to eliminate the aluminium content in circulation at the onset of the inflammatory process. Since the half-life of these proteins corresponds to about 2 weeks, the decrease in plasma albumins becomes evident long after the onset of phlogosis, unless said phlogosis is associated with a severe protein loss through the endothelia damaged by the inflammatory process (e.g. abundant intracavitary effusions): in this case the decrease of the albumins can be conspicuous and rapid.

The diagnostic importance of hypoalbuminaemia as a negative APP becomes clear only when all possible causes of reduced production (e.g.: fasting, malabsorption, liver failure) or increased loss of albumin (e.g. protein-losing enteropathies, protein-losing nephropathies) have been excluded (see also electrophoresis of the serum proteins).

Albumin is usually measured with spectrophotometers, using the bromocresol green method. However, a more accurate calculation is based on the total protein concentration and the percentage of albumin detected by electrophoresis of the serum proteins.

Transferrin (Tfr): transferrin is a negative acute phase protein, the primary function of which is to transport iron so that it can be released rapidly into the tissues. Since iron is essential for bacterial metabolism, during inflammatory states the body tends to reduce the sideremic content that might be used by pathogens. One of the mechanisms by which this result is achieved is via the reduction in the synthesis of Tfr, in order to reduce the amount of iron transported into the circulation. Similarly to albumin, however, transferrin is a "slow" negative APP: its concentration in the blood decreases within about one week. Given its central role in iron metabolism, transferrin is widely used in clinical practice in assessing the iron profile, as the integrated assessment of blood concentrations of iron, transferrin (determined as total iron binding capacity, see below), and ferritin may make it possible to differentiate primary forms of failure from inflammatory states, in which iron is present but is largely unavailable.

The most commonly used method for measuring Tfr is the assessment of the total iron binding capacity, i.e. the ability of the sample to transport iron, which is ultimately dependent on the Tfr. Excess iron is added to the sample being examined, so that it can bind to the Tfr present: the excess iron is then precipitated (e.g. by adding magnesium carbonate) and a standard spectrophotometer is used to measure the concentration of iron in the sample, which will be proportional to the amount of Tfr present.

Transport proteins of hormones and vitamins (retinol binding protein, transthyretin, cortisol binding protein): there are a series of negative acute phase proteins identified in humans, but not in all animal species, which carry hormones and vitamins: in case of inflammation, their synthesis decreases to allow an increase in the free portion of the molecules being transported. Since only the free portion of hormones and vitamins is biologically active, the decrease in the plasma concentration of these transport proteins makes it possible to increase the action of hormones and vitamins even without increasing their synthesis.

The determination of these molecules requires special, often immunologically based methods, whose use in veterinary medicine is limited by the scarcity of species-specific reagents available.

Apolipoprotein A1: apolipoprotein A1 is the protein portion of certain lipoproteins and can act, in some species, as a negative acute phase protein. Apolipoprotein A1 is obviously involved in lipid metabolism and helps to form high density lipoproteins (HDL). In humans, it is thought to also play an anti-atheromatous role. In pigs it acts as a negative acute phase protein and its plasma concentration decreases very rapidly, unlike that of the other negative APPs mentioned above. It is determined with an ELISA species-specific assay which is commercially available.

Suggested readings

1. Bochsler, P.N., Slauson, D.O., 2002. Inflammation and Repair of Tissue. In: Mechanism of Disease. A Textbook of Comparative General Pathology, third ed. Mosby, St. Louis, pp. 140–245.
2. Ceron, J.J., Eckersall, P.D., Martinez-Subiela, S., 2005. Acute phase proteins in dogs and cats: current knowledge and future perspectives. Veterinary Clinical Pathology 34, 85–99.
3. Gruys, E., Toussaint, M.J.M., Upragarin, N., Van Ederen, A.M., Adewuyi, A.A., Candiani, D., Nguyen, T.K.A., Sabeckiene (Balciute), J., 2005. Acute phase reactants, challenge in the near future of animal production and veterinary medicine. Journal of Zhejiang University Science 6B, 941–947.
4. Murata, H., Shimada, N., Yoshioka, M., 2004. Current research on acute phase proteins in veterinary diagnosis: An overview. The Veterinary Journal 168, 28–40.
5. Petersen, H.H., Nielsen, J.P., Heegaard, P.M.H., 2004. Application of acute phase protein measurements in veterinary clinical chemistry. Veterinary Research 35, 163–187.