Plasma is the non-corpuscular part of blood. It is obtained by centrifuging a sample of whole blood with an anticoagulant. In the test tube the corpuscular part will be arranged on the bottom, the plasma on the surface. If the blood is allowed to clot and centrifuged, serum is obtained. The difference between plasma and serum is that plasma does not contain fibrinogen (substance used during coagulation). Plasma contains more than 300 different proteins (pps). Many pathological conditions affect the level of pps. They are mainly synthesized in the liver. Some are produced at other sites. A normal adult contains about 70 g/L of pps. 

Functions

Many plasma proteins (PPT) are buffers, i.e. they help to keep the pH of the blood constant. They have a role in coagulation and fibrinolysis (thrombin and plasmin etc); they maintain the oncotic pressure of plasma (albumin); they have transport functions (albumin, prealbumin, globulins); they have defense functions (immunoglobulins and complement); they have enzymatic properties, many plasma proteins are proteases, some are protease inhibitors.

PPT measurement

Measurement of blood proteins occurs in serum. Quantitative measurement of a specific protein is done by chemical or immunological reactions. A semiquantitative measurement is obtained by electrophoresis: proteins are separated according to their electrical charge in electrophoresis, five separate bands of proteins have been observed, these bands change in disease. 

For diagnostic purposes, plasma proteins are separated and determined by electrophoresis on cellulose acetate or agarose gel plates or on capillary columns, at pH 8.6. This pH is higher than their isoelectric point so all proteins take on a negative charge and migrate to the anode. 

Types of plasma proteins

The main types of plasma proteins are: Prealbumin, Albumin, α1-Globulins(a1- antitrypsin, α-fetoprotein), α2-Globulins(Ceruloplasmin, haptoglobin), β-Globulins (CRP, transferrin, β2-microglobulin,) and the γ- Globulins. 

• Prealbumin, or transthyretin, precedes peak albumin and is a plasma transporter for thyroid hormones and retinol (vitamin A). Retinol acts as a co-transporter. It not only carries these hormones to the peripheral organs but also to the CNS. It migrates faster than albumin in electrophoresis and is separated by immunoelectrophoresis. It has lower levels in liver disease, nephrotic syndrome, inflammatory response in the acute phase, malnutrition. It has a short half-life (2 days). It is composed of a very high percentage of essential amino acids. This makes it a marker of good nutritional health. It helps us understand if there are diet-related problems or an absorption problem. It decreases rapidly in cases of increased catabolism.
• Albumin is the most abundant plasma protein (~40 g/L) in the normal adult. It accounts for 60% of plasma protein. It is synthesized in the liver as preproalbumin and secreted as albumin. It has a half-life in plasma of 20 days. It decreases rapidly under conditions of poor nutrition, in injury, infection, and surgery. Its main function is to maintain oncotic pressure in the blood. Oncotic pressure is the osmotic pressure exerted by plasma proteins that draws water into the circulatory system. It maintains the distribution of fluids in and out of cells and plasma volume. Eighty percent of plasma oncotic pressure is maintained by albumin. If we measure plasma proteins in a subject with lower extremity edema, we will find that they are more or less constant but the amount of albumin is definitely lower. A 10% decrease (from 4g to 3.7g) is enough to give rise to edematous manifestations.   Albumin is also a non-specific transporter of hormones, calcium, free fatty acids, drugs. It also transports bilirubin, a derivative product of hemoglobin, synthesized in the spleen, marrow, and liver. Bilirubin is absolutely insoluble and has the tendency to cross membranes, and if this happens at the encephalic level it can cause serious damage. Tissue cells can take up albumin by pinocytosis, where it is hydrolyzed into amino acids. It is useful in the treatment of liver disease, hemorrhage, shock, and burns.

Hypoalbuminemia:

Decreased albumin synthesis (liver cirrhosis, malnutrition)

Increased albumin loss: may be caused by increased catabolism in infections, or excessive excretion by the kidneys (nephrotic syndrome) or excessive loss in the intestines or severe burns (plasma loss in the absence of a skin barrier). 

The effects of hypoalbuminenia are: 

• edema due to low oncotic pressure where albumin level decreases in liver disease causing low oncotic pressure; fluid moves into interstitial spaces causing edema; 
• reduced transport of drugs and other substances in plasma; reduced protein-bound calcium; 
• decrease in total plasma calcium level; ionized calcium level may remain normal. 

Hyperalbuminemia

There are no known clinical conditions that cause the liver to produce large amounts of albumin. The only cause of hyperalbuminemia is dehydration.

• α1-Globuline
• a1-Antitrypsin is synthesized by the liver and macrophages and is released in inflammation. It is an acute phase protein. It inhibits serine proteases, which are produced endogenously such as trypsin, chymotrypsin, and hence the name, but in plasma it inhibits extracellular ones produced by leukocytes against bacteria, such as elastase. Infections lead to the release of proteases by bacteria and leukocytes. 

More than 30 types of a1-Antitrypsin are known. The most common is the M-type. 

There is a genetic deficiency of a1-Antitrypsin, which causes a 1-Antitrypsin deficiency. Altered synthesis of a1- Antitrypsin occurs in the liver but the protein is not secreted. a 1-Antitrypsin accumulates in hepatocytes and there is a deficiency in plasma. The mutation causes a failure to fold the protein into the endoplasmic reticulum of the hepatocyte, the protein is not secreted and accumulates in the hepatocyte. This accumulation activates ER stress proteins that trigger apoptosis. There is liver damage and there is a deficit in the plasma.

As clinical consequences: neonatal jaundice with cholestasis, liver cirrhosis of childhood, and pulmonary emphysema in young adults Laboratory diagnosis is based on the absence of the a1-globulin band in protein electrophoresis. We quantitatively measure a1- Antitrypsin by radial immunodiffusion, two-dimensional electrophoresis, or nephelometry. 

A1-Antitrypsin is also an anti-protease, a leukocyte elastase inhibitor that degrades elastin in the pulmonary alveoli. Excess elastase digests elastic fibers and other proteins in the connective tissue of the lungs. Individuals with pulmonary emphysema must breathe much more vigorously than normal individuals to exchange the same volume of air; their alveoli are much less elastic than normal.

a Fetoprotein (AFP) Synthesized in the embryo and fetus from liver parenchymal cells AFP levels increase gradually during intra-uterine life and reach levels (very low however) of the adult at birth.

Function is unknown in the adult; may protect the fetus from immunological attack by the mother High maternal AFP levels are associated with:

Neural tube defects (spina bifida), anencephaly (leaks into amniotic fluid). Decreased maternal AFP levels are associated with increased risk of Down syndrome In adults AFP is a tumor marker for: hepatoma and testicular cancer.

• α2-Globuline

Ceruloplasmin is synthesized by the liver, binds copper. Contains >90% serum copper. It constitutes a copper reservoir. Has oxidoreductase activity, inactivates ROS causing tissue damage in acute phase response. Important for the absorption of iron from the intestine

In Wilson's disease (a rare disease) there is an accumulation of copper in the liver and brain, associated with low plasma levels of ceruloplasmin. Copper can induce damage so a low copper diet must be done. At the intestinal level, we have a transporter that draws copper in, brings it into the enterocytes. Here there is an ATPase, Menkes' ATPase (a Cu-ATPase) that pumps copper to the circulatory system: in this way we introduce copper into the bloodstream. Copper travels mostly bound to albumin and transcuprein. When it gets to the liver, probably a reductase (as yet unidentified) reduces the copper and, at this point, the copper can enter the hepatocytes.

Copper that has arrived in the liver can follow two routes:

Bind to ceruloplasmin and return to the circulation.

End up in the bile: this is the only way the human body eliminates copper. These two steps (either copper incorporated into ceruloplasmin or copper into bile) require activity of another ATPase, Wilson's ATPase. If Wilson's ATPase is missing, the human body cannot produce ceruloplasmin.

Haptoglobin: is synthesized by the liver, binds to free hemoglobin to form complexes that are metabolized in the endothelial reticulum system (RES). Limits iron loss because it prevents Hb loss in the kidney. Plasma levels decrease during hemolysis.

• β-Globuline

Transferrin. The major iron transporter protein in plasma. 30% saturated with iron. Plasma levels drop in malnutrition, liver disease, inflammation, tumors. Iron deficiency results in increased hepatic synthesis of transferrin. It is a negative acute phase protein. 

b2 microglobulin. Component of the HLA System. Present on the surface of lymphocytes and most nucleated cells. Filtered by renal glomeruli because of its small size but for the most part (more than 99%) is reabsorbed. We find high serum levels in renal failure. In some diseases we can have an overproduction. It can be used as a tumor marker for: Leukemia, lymphoma, multiple myeloma. 

C-reactive protein (CRP). Classic example of an acute phase protein synthesized by the liver. Binds a particular pattern on the surface of microorganisms. It acts as a bridge between foreign element and effector cell. C-reactive proteins are opsonins, they promote interaction with immune system cells and phagocytosis. C-reactive protein is used as an inflammatory marker. Its dosage is assessed after surgery. Those who have had surgery are monitored. It should decrease as the surgical portion resolves. During infection in the first few days post-surgery, there is a violent increase in protein C. This is important for phagocytosis. In many inflammatory conditions such as rheumatoid arthritis we can have high plasma levels. It can also be used as a marker of cardiac ischemia.

Hypergammaglobulinemia

It can be activated by B-cell stimulation (polyclonal hypergammaglobulinemia) or by monoclonal proliferation (Paraproteinemia).

Polyclonal hypergammaglobulinemia: stimulation of many B-cell clones, produces a broad spectrum of antibodies. Gamma-globulin band appears large on electrophoresis.

Clinical conditions: acute and chronic infections, autoimmune diseases, chronic liver disease.

Monoclonal hypergammaglobulinemia: proliferation of a single clone of B cells producing a single type of Ig. Appears as a separate dense band (paraprotein or M-band) on electrophoresis. Paraproteins are characteristic of malignant B-cell proliferation.

 Clinical Condition: multiple myeloma. 

Positive Acute Phase Protein

Proteins that vary in quantity in response to inflammatory processes. They are produced by the liver and their synthesis is regulated by cytokines. They are the immediate acute response of the liver to local or systemic inflammatory processes. They have a very high increase within 24h, they are optimal markers. Following inflammation, white blood cells produce a whole series of cytokines, interleukins, etc. that serve to prepare the human body to defend itself against the inflammatory process. Among these there is IL-6 whose main task is to stimulate the liver to produce a whole series of serum proteins that serve to cope with the inflammatory state (protein C, fibrinogen, ceruloplasmin etc.). So in the presence of an inflammatory state, the liver sets about producing more serum protein. To compensate for this increased synthesis, some proteins are produced in lesser amounts, especially albumin (negative acute phase protein). 

This increase in protein production is responsible for the increase in ESR (erythrocyte sedimentation rate). For many years ESR was the classic marker of inflammatory processes, now replaced by the C-reactive protein assay that is more specific and produces fewer false positives. Acute phase plasma protein levels increase in infection, inflammation, tumors, trauma, surgery. These proteins are the acute phase reactants and are synthesized as a response to damage to the body. Examples: a1-Antitrypsin, haptoglobin, ceruloplasmin, fibrinogen, c-reactive protein

Mediators that cause these proteins to increase after injury include: leCytokines (IL-1, IL-6), tumor necrosis factors a and b, interferons, platelet activating factor.

Acute Phase Negative Protein

These proteins decrease in inflammation the values of albumin, prealbumin, transferrin.

They are mediated by the inflammatory response via cytokines and hormones.

Synthesis of these proteins decreases to conserve amino acids for positive acute phase proteins.