Plasma protein

Metamorphosis of the plasma protein of the chicken throughout the phases of embryogenesis and postembryonic development has been investigated very thoroughly (Weller and Schechtman, 1962). 

A typical picture of these changes, which also reveals most distinctly the behavior of the albumins, is shown in Fig. 71. In mammals the morphogenetic spectrum of the plasma proteins has been studied most fully in rodents (rats, mice) (Shmerling and Uspenskaya, 1955 Pantelouris and Hall, 1962) and in man (Tatarinov et al., 1963 Woods et al., 1961). In this case also, albumins did not appear until about halfway through embryogenesis, whereas certain embryonic proteins were absent from adults. 

Protein Concentration in plasma (mg/dL) Molecular weight Electrophoretic mobility at pH of 8.6 Function 

Prothrombin 150 72,000 /3-Globulin plasmin, which lyses fibrin Activated in 

Complement 100-200 /3-Globulin thrombin, which clots fibrinogen Enters into immune 

Fibrinogen 200-450 340,000 /3-Globulin reactions Clots to give fibrin 

Albumin also acts as a transport medium for a variety of substances. It is the principal, if not the only, means of transport for free bilirubin (breakdown product of heme) and free fatty acids. It also binds calcium ions, hematin, steroids, thyroxine, and various drugs and dyes. 

Owen (02) has recently reviewed the literature describing plasma protein changes following injury. Some of the salient features will be considered here. 

Since these earlier observations were made the whole technique of plasma protein estimation has considerably improved and the earlier findings substantially confirmed, namely, that after major surgery in man or after accidental injury or burning the plasma albumin falls (B20, D4, L2, Ml, M5, P5, R6, S13a). The fall, according to Owen (02), averages about 0.8 g/KK) ml (about 20 of the normal plasma albumin level), but decreases of twice this amount have been found in individual patients with the depression maximal between 4 and 10 days and sometimes normal values may not be regained for several weeks. The fall is particularly marked after extensive burns. Davis et al. (Dl) have noted an 

a-Globulins. An increase in plasma tti- and 2-globulins occurs after surgery and after burns and in the case of animals is much reduced if the animals are first starved (B12). Ceruloplasmin rises steadily after injury, and raised levels may be found a fortnight after surgery (Ml). Haptoglobulin rises until about the fourth day, with peak between 3 and 7 days, then starts to fall (02). 

A slow moving aa-globulin— variously designated aa-acute phase (AP) or a-2-glycoprotein (GP)— which is not normally detectable in the plasma of patients and experimental animals, appears in acute inflammatory states and after injury in general. It appears in the plasma 12-18 hours after the injury and remains detectable for 7-10 days or longer. Adrenalectomy prevents its appearance after injury. It can be restored in the adrenalectomized rat by corticosterone or cortisol. [For review see (BIO, 02, W8, W9).] 

There is a linearity of response to graded cortisol treatment in traumatized adrenalectomized animals (Bll). The adrenal cortical control of the appearance of slow a2-globulin is incomplete in the 2-day postpartum maternal rat, because the globulin can be demonstrated in most adrenalectomized animals in this condition. Adrenal cortical control over the appearance of the globulin is not simply in the postpartum maternal animal, because the percentage of adrenalectomized rats with demonstrable slow t2-globulin can be increased by the administration of corticosterone (H7). 

Quantitatively, proteins are the most important part of the soluble components of the blood plasma. With concentrations of between 60 and 80 g L they constitute approximately 4% of the body s total protein. Their tasks include transport, regulation of the water balance, hemostasis, and defense against pathogens. 

Some 100 different proteins occur in human blood plasma. Based on their behavior during electrophoresis (see below), they are broadly divided into five fractions albumins and ai-, tt2-, P- and y-globulins. Historically, the distinction between the albumins and globulins was based on differences in the proteins solubility -albumins are soluble in pure water, whereas globulins only dissolve in the presence of salts. 

The most frequent protein in the plasma, at around 45 g is albumin. Due to its high concentration, it plays a crucial role in maintaining the blood s colloid osmotic pressure and represents an important amino acid reserve for the body. Albumin has binding sites for apolar substances and therefore functions as a transport protein for long-chain fatty acids, bilirubin, drugs, and some steroid hormones and vitamins. In addition, serum albumin binds Ca and Mg ions. It is the only important plasma protein that is not glycosylated. 

The albumin fraction also includes transthyretin (prealbumin), which together with other proteins transports the hormone thyroxine and its metabolites. 

The table also lists important globulins in blood plasma, with their mass and function. The a- and p-globulins are involved in the transport of lipids (lipoproteins see p. 278), hormones, vitamins, and metal ions. In addition, they provide coagulation factors, protease inhibitors, and the proteins of the complement system (see p. 298). Soluble antibodies (immunoglobulins see p. 300) make up the y-globulin fraction. 

Heparinoids and mucopolysaccharides react with, and modify, many of the plasma proteins. Heparin combines with fibrinogen, globulins and albumin. As judged by electrophoresis and various types of analysis and staining, the particular plasma protein components with which heparin combines are dependent upon the concentration of protein, concentration of heparin, pH value, and salts present. This explains the somewhat contradictory statements in literature about combinations of heparin with plasma proteins. The combination may result in change of solubility of the protein and reverse protein tests . Heparin can modify the murexide reaction for calcium in serum by affecting the calcium-protein-heparin complex. Many heparinoids 

Immunoglobulins.—Crystallographic studies of immunoglobulins are under way in a number of laboratories. These include studies of complete immunoglobulin molecules, of Bence-Jones proteins, and of fragments. 

The X-ray data extend to about 4.5 A resolution, but their quality is low outside the 6 A sphere. The extreme sensitivity of the crystals of inununo-globulin to X-radiation presented considerable difficulties and about eight 

These same immunoglobulin crystals have also been the subject of a study in the electron microscope. The molecule in projection appears to have a shape varying between a Y and a T, in agreement with previous electron microscope studies and the X-ray diffraction investigation. 

Edmundson et alP have also studied crystals of the A-type Bence-Jones protein (the light chain of the immunoglobulin) from the same myeloma patient. The protein crystallizes in orthorhombic form from deionized water and as a trigonal form from ammonium sulphate. The orthorhombic form of space group P2i2i2 contains the M-S-S-M dimer. As with the euglobulin the salt-free crystals dissolved in most heavy-atom solutions, but PtCl made the crystals less soluble after which they could be treated by dilute solutions of heavy-atom salts. The rotation function has demon- 

AT-Ray studies of a second Bence-Jones protein of antigenic type (k) are being carried out.  

The proteins most amenable to routine laboratory evaluation die those in blood, urine, CSF amniotic fluid, saliva, feces, and peritoneal or pleural fluids. With few exceptions, the proteins found in all of these are derived from blood plasma. The following discussion is limited to (1) the most abundant plasma proteins, (2) changes of their concentrations in the most accessible body fluids, and (3) a few of the analytical techniques used to measure them. 

Most plasma proteins, with the exception of immunoglobulins and protein hormones, are synthesized primarily in the liver. They are secreted by the hepatocytes 

Rates of hepatic synthesis of many plasma proteins are affected by a patient s endocrine status. The effects of some steroid hormones on individual plasma protein levels are given in Table 20-5. The plasma protein levels characteristic of a specific disease may therefore be complicated by the steroid status of a patient and by an inflammatory acute phase reaction. The abnormal steroid status may be the result of an intrinsic hormonal disorder or of treatment with steroid hormones, as in inflammation. 

Several of the proteins in Table 20-3 are acute phase reactants (APR) with the concentrations of tti-antitrypsin (AAT), aj-acid glycoprotein (AAG), haptoglobin (Hp), ceruloplasmin, C4, C3, procalcitonin (PCT), and serum amyloid A (SAA) increasing in most forms of inflammation these are called positive APR. Others, such as transthyretin 

Region Protein Haif-Life pi (daltons) Analysis Method Comments 

Heparin has been reported to complex with a variety of basic species, including biogenic amines and drugs for reviews, see Refs. 10 and 391. For its possible relevance to the pharmacological properties of heparin and complexed species, mention is made here of complexes with hista-mine and anthracycline antibiotics. C.d. studies on the interaction of basic homopolypeptides with heparin and other glycosaminogly-cans have shown that heparin is able to induce an ordered, helical conformation in the polypeptide. Similar, and even more dramatic, effects were observed with mixed basic polypeptides, presumed to represent better models for the biologically relevant interactions with plasma proteins.  

Essentially as a result of its ability to bind to basic sites, heparin interacts with many proteins. Although most of these interactions (such as that with protamine, a basic protein frequently used to neutralize heparin ) are probably not of biological significance, binding to plasma proteins and to proteins exposed on the surface of endothelial cells has an important influence on the circulation system. 

The fluidity of blood is a result of the inhibition of a complex series of enzymic reactions in the coagulation cascade (see Fig. 10). When triggered either intrinsically (by contact with foreign surfaces ), or extrinsically (by tissue factors from damaged cells), inactive proenzymes (factors XII, XI, IX, and X) are transformed into activated pro-teinases (Xlla, XIa, IXa, and Xa, respectively). Each proteinase catalyzes the activation of the following proenzyme in the sequence, up to formation of thrombin (Factor Ila), another proteinase that catalyzes partial 

— Simplified Representation of the Coagulation Cascade. [AT denotes Antithrombin segments represent exogenous heparin, where is a segment 

Heparin can affect various steps of the coagulation cascade. It is now generally recognized that heparin acts as an anticoagulant of blood mainly by dramatically potentiating the inhibitory eflfect of antithrombin on the proteinases. However, heparin can also interact directly with some of the proteinases, modulate most of the reactions in the cascade by interaction with such co-factors as Ca ions and phospholipids, and significantly affect other functions (such as the platelet function) or processes (such as fibrinolysis) associated with formation and lysis of 

Complexation of antithrombin with thrombin (and probably also with other proteinases) occurs by formation of a covalent bond which, by subsequent splitting, produces unaltered proteinase and irreversibly modified antithrombin.405,406 The strength of heparin-antithrombin complexes, as determined by u.v.-difference,405 fluorescence,406,407 af- 

A heparin-chain segment longer than its minimal binding site (the pentasaccharide 12) is necessary for enhancing the antithrombin-me- 

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Golander C G, Lin Y S, Fllady V and Andrade J D 1990 Wetting and plasma-protein adsorption studies using surfaoes with a hydrophobieity gradient Colloids Surf. 49 289-302... 

Kurrat R, Wallvaara B, Marti A, Textor M, Tengvall P, Ramsden J J and Spencer N D 1998 Plasma protein adsorption on titanium Colloids Surf. B 11 187-201... 

Affinity chromatography is used in the preparation of more highly purified Factor IX concentrates (53—55) as well as in the preparation of products such as antithrombin III [9000-94-6] (56,57). Heparin [9005-49-6], a sulfated polysaccharide (58), is the ligand used most commonly in these appHcations because it possesses specific binding sites for a number of plasma proteins (59,60). 

Fig. 6. Share of U.S. market occupied by human albumin/plasma protein fraction, (—) Factor VIII concentrate, (-) intravenous immunoglobulin...

Table 9. Properties of Human Albumin and Plasma Protein Fraction ...

Characteristic Human albumin Plasma protein fraction ... 

Mifepristone. After oral adininistration, peak plasma levels of mifepristone (84) (RU 486) are reached in 1 h and over 95% was bound to plasma proteins (351,352). The plasma half-life of RU 486 is approximately 24 h (352,353). In humans, monodemethylated (98), didemethylated (99) and alcohoHc nondemethylated (100) metaboHtes of RU 486 have been identified (351). These metaboHtes show some progestin-binding affinity, approximately five to ten times lower than that of RU 486 itself. RU 486 and its metaboHtes can be measured by radioimmunoassay and hplc (353,354). 

Care should be exercised when attempting to interpret in vivo pharmacological data in terms of specific chemical—biological interactions for a series of asymmetric compounds, particularly when this interaction is the only parameter considered in the analysis (10). It is important to recognize that the observed difference in activity between optical antipodes is not simply a result of the association of the compound with an enzyme or receptor target. Enantiomers differ in absorption rates across membranes, especially where active transport mechanisms are involved (11). They bind with different affinities to plasma proteins (12) and undergo alternative metaboHc and detoxification processes (13). This ultimately leads to one enantiomer being more available to produce a therapeutic effect. 

Materials may be absorbed by a variety of mechanisms. Depending on the nature of the material and the site of absorption, there may be passive diffusion, filtration processes, faciHtated diffusion, active transport and the formation of microvesicles for the cell membrane (pinocytosis) (61). EoUowing absorption, materials are transported in the circulation either free or bound to constituents such as plasma proteins or blood cells. The degree of binding of the absorbed material may influence the availabiHty of the material to tissue, or limit its elimination from the body (excretion). After passing from plasma to tissues, materials may have a variety of effects and fates, including no effect on the tissue, production of injury, biochemical conversion (metaboli2ed or biotransformed), or excretion (eg, from liver and kidney). 

Uranium can enter the human body orally, by inhalation, and through the skin and mucous membranes. Uranium compounds, both soluble and insoluble, ate absorbed most readily from the lungs. In the blood of exposed animals, uranium occurs in two forms in equiUbrium with each other as a nondiffusible complex with plasma proteins and as a diffusible bicarbonate complex (242). 

Purification. Hemoglobin is provided by the red blood ceU in highly purified form. However, the red ceU contains many enzymes and other proteins, and red ceU membranes contain many components that could potentially cause toxicity problems. Furthermore, plasma proteins and other components could cause toxic reactions in recipients of hemoglobin preparations. The chemical modification reactions discussed herein are not specific for hemoglobin and may modify other proteins as well. Indeed, multifimctional reagents could actually couple hemoglobin to nonhemoglobin proteins. 

The hemorrhagic diathesis in patients with coagulation disorders is because of either an abnormaUty of one or more plasma proteins and/or platelets necessary for normal blood coagulation or the spontaneous presence of a circulating anticoagulant. Specific laboratory techniques are required for the precise identification of these disorders. 

Procainamide may be adininistered by iv, intramuscular (im), or po routes. After po dosing, 75—90% of the dmg is absorbed from the GI tract. About 25% of the amount absorbed undergoes first-pass metaboHsm in the fiver. The primary metabolite is A/-acetylprocainamide (NAPA) which has almost the same antiarrhythmic activity as procainamide. This is significant because the plasma concentration of NAPA relative to that of procainamide is 0.5—2.5. In terms of dmg metabolism there are two groups of patients those that rapidly acetylate and those that slowly acetylate procainamide. About 15—20% of the dmg is bound to plasma proteins. Peak plasma concentrations are achieved in 60—90 min. Therapeutic plasma concentrations are 4—10 lg/mL. Plasma half-lives of procainamide and NAPA, which are excreted mainly by the kidneys, are 2.5—4.5 and 6 h, respectively. About 50—60% is excreted as unchanged procainamide (1,2). 

Disopyr mide. Disopyramide phosphate, a phenylacetamide analogue, is a racemic mixture. The dmg can be adininistered po or iv and is useful in the treatment of ventricular and supraventricular arrhythmias (1,2). After po administration, absorption is rapid and nearly complete (83%). Binding to plasma protein is concentration-dependent (35—95%), but at therapeutic concentrations of 2—4 lg/mL, about 50% is protein-bound. Peak plasma concentrations are achieved in 0.5—3 h. The dmg is metabolized in the fiver to a mono-AJ-dealkylated product that has antiarrhythmic activity. The elimination half-life of the dmg is 4—10 h. About 80% of the dose is excreted by the kidneys, 50% is unchanged and 50% as metabolites 15% is excreted into the bile (1,2). 

Phenytoin s absorption is slow and variable yet almost complete absorption eventually occurs after po dosing. More than 90% of the dmg is bound to plasma protein. Peak plasma concentrations are achieved in 1.5—3 h. Therapeutic plasma concentrations are 10—20 lg/mL but using fixed po doses, steady-state levels are achieved in 7—10 days. Phenytoin is metabolized in the fiver to inactive metabolites. The plasma half-life is approximately 22 h. Phenytoin is excreted primarily in the urine as inactive metabolites and <5% as unchanged dmg. It is also eliminated in the feces and in breast milk (1,2). Prolonged po use of phenytoin may result in hirsutism, gingival hyperplasia, and hypersensitivity reactions evidenced by skin rashes, blood dyscrasias, etc... 

EoUowing po administration moricizine is completely absorbed from the GI tract. The dmg undergoes considerable first-pass hepatic metabolism so that only 30—40% of the dose is bioavailable. Moricizine is extensively (95%) bound to plasma protein, mainly albumin and a -acid glycoprotein. The time to peak plasma concentrations is 0.42—3.90 h. Therapeutic concentrations are 0.06—3.00 ]l/niL. Using radiolabeled moricizine, more than 30 metabolites have been noted but only 12 have been identified. Eight appear in urine. The sulfoxide metabolite is equipotent to the parent compound as an antiarrhythmic. Elimination half-life is 2—6 h for the unchanged dmg and known metabolites, and 84 h for total radioactivity of the labeled dmg (1,2). 

Tocainide is rapidly and well absorbed from the GI tract and undergoes very fitde hepatic first-pass metabolism. Unlike lidocaine which is - 30% bioavailable, tocainide s availability approaches 100% of the administered dose. Eood delays absorption and decreases plasma levels but does not affect bio availability. Less than 10% of the dmg is bound to plasma proteins. Therapeutic plasma concentrations are 3—9 jig/mL. Toxic plasma levels are >10 fig/mL. Peak plasma concentrations are achieved in 0.5—2 h. About 30—40% of tocainide is metabolized in the fiver by deamination and glucuronidation to inactive metabolites. The metabolism is stereoselective and the steady-state plasma concentration of the (3)-(—) enantiomer is about four times that of the (R)-(+) enantiomer. About 50% of the tocainide dose is efirninated by the kidneys unchanged, and the rest is efirninated as metabolites. The elimination half-life of tocainide is about 15 h, and is prolonged in patients with renal disease (1,2,23). 

Elecainide is weU absorbed and 90% of the po dose is bioavailable. Binding to plasma protein is only 40% and peak plasma concentrations are attained in about 1—6 h. Three to five days may be requited to attain steady-state plasma concentrations when multiple doses are used. Therapeutic plasma concentrations are 0.2—1.0 lg/mL. Elecainide has an elimination half-life of 12—27 h, allowing twice a day dosing. The plasma half-life is increased in patients with renal failure or low cardiac outputs. About 70% of the flecainide in plasma is metabolized by the Hver to two principal metaboUtes. The antiarrhythmic potency of the meta-O-dealkylated metaboUte and the meta-O-dealkylated lactam, relative to that of flecainide is 50 and 10%, respectively. The plasma concentrations of the two metaboUtes relative to that of flecainide are 3—25%. Elecainide is mainly excreted by the kidneys, 30% unchanged, the rest as metaboUtes or conjugates about 5% is excreted in the feces (1,2). 

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