TRACKING DYSPROTEINAEMIA IN THERMAL INJURIES USING SERUM PROTEIN ELECTROPHORESIS

Wedler V., Prokop S., Künzi W., Meyer V.E., Stocker R., Bürgî U.

Burns Centre, Clinic for Reconstructive Surgery, University Hospital, Zurich, Switzerland

SUMMARY. Serum protein electrophoresis is a routine method for diagnostics of dyslipoproteinaemia. The last 25 years' literature gives an extensive survey of the technical possibilities of separating total plasma protein qualitatively and quantitatively in its fractions. Although there are specific descriptions of dysproteinaemia for a multitude of acute and chronic diseases, its course in thermal injuries has been little described. From February to October 1997 serum protein electrophoresis was performed prospectively in 24 patients suffering from burn injuries, with total body surface ranging from 15 to 72% (mean: 30%). They were followed from the acute phase until discharge from the intensive care unit. The individual fractions were analysed and compared with each other. A typical curve with a compensatory shift to the alpha-1 and alpha-2 fractions was demonstrated in serum protein electrophoresis of the investigated burn patients. It is not sufficient to analyse only total serum protein and alburnin concentration to control hepatic protein synthesis. Analysis of the other protein fractions and different serum proteins (alpha-1 acid glycoprotein, C-reactive-protein, high-density lipoproteins, low-density lipoproteins, caeroluplasmin and transferrin) warrants consideration to prevent insufficient substitution of alburnin with the possible risk of decreasing endogenous protein synthesis.

Introduction

Serum protein electrophoresis is a routine method used for diagnosing dysproteinaerma. The technical background to the qualitative and quantitative separation of plasma proteins into individual fractions has been widely covered in the literature of the last 25 years. Specific kinds of dysproteinaemia have also been described for a large number of acute and chronic illnesses. Dysproteinaemia in burn victims has, however, received only scant attention.
Between February and October 1997, we documented the results of serum protein electrophoresis carried out on samples from 24 burn patients with TBSA between 15% and 72% (average: 30%). Samples were taken throughout the patients' stay in intensive care.
In the phase immediately following a burn injury there is a well-known and characteristic fall in total plasma protein, caused primarily by the massive loss of alburnin. Standard clinical practice usually involves the subsequent measurement of total protein and alburnin levels, both of which can remain depressed for days or even weeks despite adequate patient nutrition. This research shows that in addition to this hypoalburninaemia and hypoprealburninaemia, there is a reorientation of protein synthesis to favour the alpha-I and alpha-2 fractions. Serum protein levels then normalize as treatment progresses, with the exception of the gamma fraction, where depressed levels in the early treatment phase are followed by a compensatory rise in the later stages. Serum protein electrophoresis enabled us to identify these qualitative changes in protein synthesis in burn victims, and made it possible to follow the typical development of these changes over the course of treatment.
Serum protein electrophoresis is a routine method used for diagnosing dysproteinaemia. Dysproteinaemia is a condition involving quantitative and qualitative changes in serum proteins and is associated with a large number of disease conditions. Serum protein electrophoresis is a tool used for identifying and monitoring changes in malignant turnouts, indicators of acute and chronic inflammation, liver diseases, antibody deficiencies, monoclonal gammopathies, etc. The heterogeneous mix of over 100 known serum proteins can be separated into four component fractions (alpha-1, alpha-2, beta and gamma globulins) (Table I) on a cellulose sheet in the electric field of the electrophoresis chamber. At the same time, the relative proportion of the total protein content represented by each individual protein can be calculated using the areas under the individual peaks of the extinction curves.

Given that the total protein content of the relevant plasma is known, these relative values can be converted to absolute values. Different authors have given different values for the concentrations of the individual proteins in blood serum. The observations made in this research are compared with reference values defined by our chemical institute (Table II).

There is a dynamic balance between protein biosynthesis, metabolization (especially in the peripheral organs), and excretion through the gastrointestinal tract. All serum proteins are synthesized in the liver, with the exception of the gamma globulins, which are produced in the beta lymphocytes. In a healthy person, the alburnin-toglobulin ratio is 1.7 to 1.
Plasma proteins have the shortest half-life of all the proteins in the body and are therefore particularly sensitive to acute or chronic changes in amino acid and protein metabolism. The direct loss of these proteins through a traumatic event (such as acute bleeding or open wounds) and/or relative loss through diffusion into extravasal space (through leaking capillaries or following organ or multiorgan failure) can have a huge impact on total protein content. Such losses result in the cessation or significant impairment of particular functions performed by the proteins of one or all serum fractions. Plasma proteins carry out a range of such functions: a nutritional function (protein reservoir), a carrier function (they have large surface areas with numerous hydrophilic and lipophilic bonding positions), maintenance of colloid-osmotic pressure (through regulation of the distribution of water between plasma and interstitium), a buffer function (constant pH), and protection from blood loss (fibrinogen).
Skin burns have major implications for alburnin loss since the skin stores between 30 and 40% of all alburnin in the body.

Materials and methods

The study was carried out between February and November 1997, using 24 patients (19 men and 5 women) at the burns centre of the Clinic for Reconstructive Surgery in Zurich University Hospital. The average TBSA of the patients was 30% (range: 15-72%) and the average age of the patients was 40.4 yr (range: 20-88 yr). The average length of stay in intensive care was 23 days (range: 3-93 days). Four of the patients died.
The patients all received surgical treatment which followed standard clinic practice: 1. bath and debridement on arrival; 2. treatment of burns, using split-skin grafts after 34 days; 3. treatment of healing wound sites, using split-skin gra s or keratinocytes as soon as the patient's clinical condition allowed. From arrival onwards, all patients received enteral gavage feeding. A serum protein electrophoresis was carried out at regular intervals of 1-2 days during the course of intensive care treatment, the latter being categorized into acute, secondary, and post-secondary phases. Average absolute values were calculated for each fraction and patient for each of these hospitalization phases. These values were then used to calculate an average absolute value for each fraction across the whole patient group. Absolute values were used when considering changes in protein levels in individual patients, and relative values were used when comparing changes across the group as a whole. Acid glycoprotein and high-density lipoproteins (HDL) (alpha- 1 fraction), caeruloplasmin (alpha-2 fraction), low-density lipoproteins (LDL), transferrin and C reactive protein (CRP) (beta globulins) were also measured at the same intervals. Further data regarding the group as a whole are given in Table III.

Total protein
All patients suffered from hypoproteinaemia during the first phase of treatment. In 17 patients this condition gradually disappeared. Fig. 1 illustrates this hypo- proteinaernia during days 1-3 and the subsequent return to normal levels.

Alburnin and prealburnin
Absolute hypoalburninaemia and relative hypoalburninaernia were observed in all patients at the beginning of treatment. Fig. 2 illustrates the deterioration in alburnin levels between days 2 and 3 of treatment. Compared with the situation regarding total protein, the return to normal levels began relatively late - from the second week of treatment onwards. Prealburnin levels remained below normal (reference) levels throughout the duration of the patients' stay in intensive care.

Alpha-1 fraction
In a compensatory reaction, the average value of the alpha-1 fraction rose steeply during the first three days of treatment to well above normal levels. This value then remained above the normal range, even though total protein levels were low, and returned to normal (reference) levels only during the second phase of treatment (Fig. 3). With the exception of one patient, HDL levels always remained within the reference range. Alpha acid protein levels were higher than normal in all patients throughout their stay in intensive care.

Alpha-2 fraction
From the third day of treatment, and despite the general hypoproteinaemia, alpha-2 fraction levels rose continuously and remained above normal values throughout treatment (Fig. 4). Caeruloplasmin stayed at normal levels in all patients at all times.

Beta fraction
Although total protein levels were lower than normal, the beta fraction remained at normal levels in 18 patients and was only slightly below normal throughout treatment in six patients (Fig. 5). There were raised levels of CRP in all patients during the acute treatment phase and at the beginning of the secondary treatment phase. LDL levels in 22 patients were below normal throughout treatment. Transferrin levels remained normal in 14 patients, but were lower than usual in 10 patients during the first two treatment phases.

Alburnin substitution simply depresses the body's own production of alburnin. Electrophoresis measurements, however, provide detailed qualitative information about increases or decreases in the synthesis of proteins in each individual fraction, information which can then be used more effectively in treatment or the planning of diets.

Gamma fraction
Gamma fraction levels were below normal in the acute treatment phase and within the reference range during the secondary treatment phase, rising above normal levels during the final treatment phase (Fig. 6).

Discussion

Changes in the constituents of the individual protein fractions and changes in the relative amounts of each fraction in the serum can be observed in many illnesses, and these changes are exploited in clinical treatment. Pathophysiological regulatory mechanisms have yet to be fully investigated in burn victims. The rapid development of dysproteinaernia at the beginning of the burn trauma, as a consequence of alburnin loss, and nutrition-related dysproteinaemia during treatment are well-known phenomena. The data from our patients showed that compensatory changes took place in all serum protein fractions and we compared these with changes in total protein levels. It was noticeable that the amount of alpha1 and alpha-2 proteins rose, even though total protein levels were lower. This general hypoproteinaemia lasted into the second week of treatment. There was also a delay before alburnin levels began to return to normal. No alburnin substitutes were given to any patients. All protein fractions fell below normal levels in days 1-3. Alpha-1 protein levels rose steeply in the first two days of treatment (even though total protein levels were depressed). Levels then returned to the upper reaches of the reference range from day 5 and remained constant throughout the remainder of treatment, even though acid glycoprotein levels in all patients were higher than normal throughout treatment. A compensatory rise in the alpha-2 fraction was observed from day 3 onwards and this continued throughout treatment, with levels sometimes rising to as much as double normal values. Despite the increase in c-reactive proteins in the first two thirds of treatment, b-fraction levels constantly remained within the reference range. The gamma fraction rose parallel to total protein levels and was at normal levels from the second treatment week onwards. After the third week of treatment, gamma fraction levels rose above normal levels, despite normoproteinaemia. We found that the results of serum protein electrophoresis in burn victims describe a typical progression. During the acute phase, inflammation parameters and proteins responsible for oxidative processes were at raised levels from day 2 posttrauma. An increase in acute phase proteins on days 6-8, as described by Moody, was not observed.

Conclusion

We have to assume that the reorganization of synthesis processes is achieved at the expense of some proteins. Increased LDL reflects an increased demand for cholesterol (for production of steroid hormones and membranes and as a key structural component of many tissue and plasma lipoproteins). If we are to understand the various compensatory mechanisms taking place within each fraction in the context of an overall hypoproteinaemia, then a range of known serum proteins will have to be measured at regular intervals over the course of recovery from burn injuries. The assumption currently made, i.e., that protein synthesis in the liver can be evaluated through measurement of alburnin and total protein (and corrected by supplying alburnin substitute), is not supported by this work. Alburnin substitution simply depresses the body's own production of alburnin. Electrophoresis measurements, however, provide detailed qualitative information about increases or decreases in the synthesis of proteins in each individual fraction, information which can then be used more effectively in treatment or in the planning of diets.

RESUME. L'électrophorèse de la protéine sérique est une méthode de routine pour le diagnostic de la dyslipoprotémémie. Depuis 25 ans la littérature présente toutes les possibilités techniques pour séparer la protéine plasmatique qualitativement et quantitativement dans ses fractions. Il y a des descriptions spécifiques de la dysprotéinémie relatives à une grande varicté de maladies aiguës et chroniques, mais le cours de la dysprotéinemie dans les brûlures a été rarement décrite. Dans la période février-octobre 1997 les Auteurs ont effectué une étude de l'électrophorèse de la protéine sérique dans 24 patients atteints de brûlure, avec une surface corporelle totale brûlée entre 15 et 72% (valeur moyenne: 30%). Les patients ont été étudiés depuis la phase aiguë jusqu'à la sortie du service de réanimation. Les fractions individuelles ont été analysées et confrontées entre elles. Une courbe typique avec un mouvement compensatoire vers les fractions alpha-1 et alpha-2 a été démontrée dans l'électrophorèse de la protéine sérique des patients brûlés étudiés. Il n'est pas suffisant d'analyser seulement la concentration de protéine et d'alburnine du sérum total pour contrôler la synthèse de la protéine hépatique. L'analyse des autres fractions protéiques (alpha-1 glycoprotéine acide, C-protéineréactive, les lipoprotéines à haute densité, la céruléoplasmine et la transferrine) mérite d'être considerée pour prévenir une substitution insuffisante de l'alburnine, avec le risque de diminuer la synthèse de la protéine endogène.

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