In the actin scavenging system, actin filaments are capped and depolymerized by gelsolin. The actin monomers are then transferred to Gc-globulin and are cleared from the circulation. The net result is that Gc-globulin is consumed. A low level can therefore act as a marker of major organ damage in conditions such as fulminant hepatic failure, acetaminophen overdose, multiple trauma and septic shock. The level of actin-free Gc-globulin indicates the actin-scavenging reserve available. If this is below a clinically determined cutoff level, the patient's prognosis will be correspondingly poor, because further actin release will lead to intravascular filament formation, microthrombi and endothelial injury, compromising the circulation and starting a vicious circle of increasing organ damage and actin release.

AntibodyShop has recently launched a rapid sandwich ELISA measuring free Gc-globulin. The rapid (<1 hour) sandwich assays is for the determination of free Gc-globulin in human serum or plasma. This makes it possible to track the free Gc-globulin reserve in critically ill patients during their clinical course, so that decisions can be made on whether to treat with plasma as a source of Gc-globulin. It can also enter into the process of decision-making on the need for acute liver transplantation.

Predictive values obtained with the Gc-globulin measurement are comparable to those obtained with the King&amp;amp;apos;s College Hospital (KCH) criteria in acute liver failure and the trauma injury and severity score (TRISS) in cases of multiple trauma.

Jack-of-all-trades - master of one?

Gc-globulin (group-specific component globulin or Gc protein) is a multifunctional plasma glycoprotein of molecular mass 51-58 kDa.1,2 While classified as an a2-globulin, it is structurally and genetically related to serum albumin, its gene being located close to the albumin gene on chromosome 4q11-q22. Its many functions can be confusing, as the protein comes up in the scientific literature in a variety of contexts. First, the occurrence of different allelic forms that can readily be detected by electrophoresis of blood traces (GC1F(fast), GC1S(slow) and GC2 in order of mobility, plus over 124 variants worldwide) made it interesting for forensic purposes before the days of DNA fingerprinting. Frequencies of Gc-variants in different populations were then related to disease prevalence, including that of diabetes mellitus. Recent surveys tend to support the view that this "genetic (as)sociology" is a red herring which has served to increase publications more than pathophysiological insight. Secondly, Gc-globulin is the major circulating vitamin D binding protein (DBP), giving it its alternative name. Here too there are doubts about the significance of its physiological or pathophysiological role. It does seem to form a reservoir and damper for 25-hydroxy-vitamin D, but Gc-globulin knockout mice live perfectly happily except for being somewhat less resistant to vitamin-D deficiency and more resistant to vitamin D intoxication.2 Thirdly, Gc-globulin can be converted by partial deglycosylation (mediated by B and T lymphocytes) into a macrophage-activating factor known as DBP-MAF or Gc-MAF.3 Not all variants can convert; for example, GC2 has a non-glycosylated Lys residue instead of the glycosylated Thr at position 420. This suggests that macrophage activation by DBP-MAF is a redundant pathway. So why then is Gc-globulin present in quite high concentration in the blood, and furthermore a protein for which congenital deficiency has not been described? Maybe the answer lies in another, this time a vitally important function, that of scavenging actin released from damaged cells.4

The actin-scavenger system5

Actin is the most abundant protein in eukaryotic cells and is released into the circulation by dead or dying cells. There it can form long filaments which may trigger intravascular coagulation if not rapidly removed. This can lead to a condition resembling multiple organ dysfunction syndrome (MODS).6 Cells from compromised organs release actin in both the polymerized fibrous (F) form and the monomer (G) form. In the actin scavenging system (see Fig. 1), F actin is depolymerized by the muscle-derived protein gelsolin, which exists in intramuscular and circulating forms. Circulating gelsolin caps the ends of the F actin filaments, binding the terminal units and also severing the filaments into monomers. G-actin monomers are then transferred to Gc-globulin, which has a single high-affinity actin binding site at the junction of its second and third domain. This binding is associated with a conformational change in the Gc-globulin, the angle between the domains being slightly altered, and does not appear to interact significantly with vitamin-D binding, which takes place in the relatively remote first domain. Transfer of actin monomers to Gc-globulin is thought to result from its higher binding affinity than that of gelsolin to the actin monomer. During the scavenging process both actin-gelsolin and actin-Gc-globulin complexes are formed, and both are cleared from the circulation much more rapidly than the free proteins, which have half-lives of about 1 (Gc-globulin) or 2 days (gelsolin). The half-life of actin-Gc-globulin in vivo is about 30 minutes. The net result is that both gelsolin and Gc-globulin are consumed. Free gelsolin can be regenerated by transfer of actin to Gc-globulin, but the actin-Gc-globulin complex does not dissociate and is cleared. What actually happens to gelsolin levels depends on the rates of formation, dissociation and clearance of actin-gelsolin complex. There are limits to how quickly gelsolin and Gc-globulin can be replaced. Gc-globulin is considered by some authors (not all) to be an acute-phase reactant whose synthesis in the liver is stimulated in certain inflammatory conditions, and even though Gc-globulin synthesis may be greatly increased, this cannot compensate for the reduction in concentration due to massive complex formation with released actin. Gelsolin is released from muscle and is not an acute-phase reactant, so that it can be depleted despite transfer of actin to Gc-globulin. The net effect on the levels of these proteins in individual patients with varying degrees of inflammation and organ damage depends on the balance between new protein synthesis and clearance of actin-protein complexes.7

The end result of the foregoing is that actin release from damaged cells reduces the circulating level of free Gc-globulin. A low level can therefore act as a marker of major organ damage such as fulminant hepatic failure,8,9 acetaminophen (paracetamol) overdose10,11 and multiple trauma.12,13 Septic shock and the organ damage associated with it may also result in reduced Gc-globulin levels14 but this has been less extensively studied. The level of actin-free Gc-globulin indicates the actin-scavenging reserve available at the time of blood sampling. If this is below a clinically determined cutoff level, the patient&amp;amp;amp;apos;s prognosis will be correspondingly poor, because exhaustion of the reserve by further actin release will lead to intravascular filament formation, microthrombi and endothelial injury, further compromising the circulation and starting a vicious circle of increasing organ damage and actin release. Because of the short half life of actin-Gc-globulin complexes, the total Gc-globulin level also correlates with actin-scavenging reserve and prognosis, if in theory rather less well than the free Gc-globulin level. For example, after acetaminophen overdose, the mean fall in the actin-free Gc-globulin level is more marked than the mean fall in the total Gc-globulin level.11,15 Up to now, most analytical methods have determined total Gc-globulin, the determination of free Gc-globulin hitherto relying on more complicated methods that are too slow to be of use in clinical decision-making.

Analysis of circulating Gc-globulin

Serum or plasma levels of Gc-globulin range from approximately 200 to 600 µg/ml in healthy donors. This makes it fairly easy to measure by simple immunochemical techniques such as immunodiffusion, turbidimetry or nephelometry using polyclonal antibodies. These techniques measure total Gc-globulin, i.e. whether or not complexed with actin. Total Gc-globulin can also be measured by inhibition-ELISA with polyclonal or monoclonal antibodies. The choice of method depends on the apparatus, reagents and experience of the laboratory concerned.

The determination of free Gc-globulin is more complex. Most published work makes use of rocket immunoelectrophoresis, whereby the height of the precipitin peaks formed on electrophoresis of sample through a gel containing polyclonal antibody is determined. A double determination is performed on each sample, before and after incubation with an excess of G-actin. The difference in peak height for the sample containing a mixture of free and actin-complexed Gc-globulin and that in which all the Gc-globulin is presumed to be complexed with actin is then used to calculate the amount of free Gc-globulin originally present in the sample, using calibrators of purified free and actin-complexed Gc-globulin. This is a slow, indirect and relatively imprecise procedure, taking more than one working day to obtain results. Free and actin-complexed Gc-globulin can also be separated by PAGE without SDS and quantified by immunoblotting.15 This is a more direct method, but subject to the imprecision of quantitative immunoblotting.

An elegant and direct way of measuring free Gc-globulin is by a rapid sandwich ELISA. This depends on capturing Gc-globulin in the sample with a coat of monoclonal antibody that is capable of binding Gc-globulin whether or not it is complexed with actin. Actin-free Gc-globulin bound to the coat is then detected with a labeled monoclonal antibody whose binding to Gc-globulin is blocked by the prior binding of actin. Thus only actin-free Gc-globulin is measured. The same principle can be extended to measuring total Gc-globulin by using a labeled detection antibody whose binding to Gc-globulin is unaffected by actin-binding, and to measuring actin-Gc-globulin complex by means of a detection antibody against an exposed part of bound actin. The principle for determining free Gc-globulin is straightforward and was first published some years ago,16 but has, surprisingly, not received the attention that it seems to merit.

AntibodyShop has recently completed development of the first of these rapid (<1 hour) sandwich assays and is launching a kit for the determination of free Gc-globulin in human serum or plasma (see Fig. 2). This makes it possible to track the free Gc-globulin reserve in critically ill patients during their clinical course, so that decisions can be made on whether to treat with plasma (as a source of Gc-globulin) or Gc-globulin concentrate, when available. It can also enter into the process of decision-making on the need for acute liver transplantation.

Interpretation of Gc-globulin concentrations

Most of the clinical studies on the prognostic value of Gc-globulin determination have determined the total Gc-globulin concentration in serum, usually in post-hoc analyses of stored samples from various groups of patients. The results have then been correlated with the outcome and used to determine predictive values in relation to determined cutoff levels. In fulminant hepatic failure, using a cutoff value of ? 100 µg/mL for serum total Gc-globulin on admission (determined by rocket immunoelectrophoresis) gave positive predictive values for patient non-survival of 100% for acetaminophen-related cases and 79% for other cases, with corresponding negative predictive values (patient survival with levels >100 µg/mL) of 53% and 60%. These predictive values are slightly better than those obtained with the King&amp;amp;apos;s College Hospital (KCH) criteria.8 In acute liver failure after acetaminophen overdose, using a cutoff value of ? 120 g/mL for serum total Gc-globulin on day 2 gave a positive predictive value for the development of hepatic encephalopathy (coma grade II) of 75%, while the negative predictive value was 91%.11 Lee et al.15 used a cutoff value of ? 34 µg/mL for serum free Gc-globulin (determined by PAGE and immunoblotting) to predict survival in fulminant hepatic failure, obtaining positive predictive values of 68% for early sera and 89% for later sera. As might be expected, the cutoff value for free Gc-globulin is considerably lower than those for total Gc-globulin. AntibodyShop is participating in a new clinical study which will serve to determine appropriate cutoffs and predictive values for its new test.

In cases of multiple trauma, using a cutoff value of ?200 µg/mL for serum total Gc-globulin on admission (determined by rocket immunoelectrophoresis) gave a positive predictive value for patient non-survival of 69% and a negative predictive value (patient survival with levels >200 µg/mL) of 84%. Sensitivity and specificity were 56% and 90%, respectively. These values are closely comparable to those obtained from the trauma injury and severity score (TRISS).12

As different analytical methods may give slightly different results, and as the basal level of Gc-globulin for a particular patient will often be unknown, the clinician will have to be guided by rough-and-ready rules of thumb, pending detailed information on the cutoff-values applicable to the laboratory method used. The 2.5 centile value for normal actin-free Gc-globulin levels as determined by the new method is about 100 µg/ml. So when free Gc-globulin levels are below 100 µg/ml, this should alert the clinician to the possibility of a process that is depleting the actin-scavenger reserve. Serial determinations will then show how the process is evolving and indicate whether plasma or appropriate plasma fractions (if available) should be given to replenish the reserve.

Indications for Gc-globulin determination

From the foregoing it is apparent that it would be reasonable to determine serum or plasma levels of Gc-globulin in the following conditions: fulminant hepatic necrosis, acetaminophen overdose, multiple trauma, rhabdomyolysis, septic shock, MODS, extensive burns, and pancreatitis; in fact, in any condition in which major organ damage is to be expected. It is hoped that the availability of rapid methods for determining Gc-globulin, and particularly free Gc-globulin, will permit an improvement in the management of patients with these conditions, so often the subject of retrospective studies.

L.O. Uttenthal

References

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14. Lee WM, Reines D, Watt GH, Cook JA, Wise WC, Halushka PV, Galbraith RM (1989) Alterations in Gc levels and complexing in septic shock. Circ Shock 28:249-255.

15. Lee WM, Galbraith RM, Watt GH, Hughes RD, McIntire DD, Hoffman BJ, Williams R (1995) Predicting survival in fulminant hepatic failure using serum Gc protein concentrations. Hepatology 21:101-105.

16. Osawa M, Erukhimov J, Galbraith RM (1994) Immunoassay of Gc globulin (vitamin D-binding protein) in complexed form with actin. Biomed Res 15:369-376.