Walter Petek

Basic fibroblast growth factor (BFGF) immunoreactivity as a possible link between head injury and impaired bone fracture healing

Healing of fractures of long bones or large joints is often accelerated in patients with severe traumatic brain injury (TBI). However, in these patients an early fracture healing is accompanied by hypertrophic callus formation or heterotopic ossifications which might even result in an ankylosis of the affected joints. It seems that enhanced osteogenesis in patients suffering from TBI could be caused by some humoral factors, since the sera of these patients strongly promote the growth of osteoblast cells in vitro. However, humoral growth promoting factors which could perhaps induce enhanced osteogenesis are not yet identified. Hence, the aim of this study was to analyse if basic fibroblast growth factor (bFGF) could be related to the phenomenon of enhanced osteogenesis, since bFGF stimulates the growth of osteoblasts in vitro and could be found both in the brain and the bone tissue. For that purpose the values of bFGF immunoreactivity were determined in the sera of patients with TBI and bone fractures (n = 8) as well as in the sera of patients with either TBI alone (n = 10) or bone fractures alone (n = 7), during a period of three months after injury. Quantification of the bFGF immunoreactivity was done using the ELISA based on monoclonal antibodies raised against the recombinant human bFGF. The bFGF immunoreactivity values obtained were also compared with the values determined in the sera of normal, healthy persons (n = 9). In the group of patients with bone fractures alone only a transient increase of bFGF immunoreactivity (threefold above the normal values) was observed in the second week after injury. A similar increase of the values of bFGF immunoreactivity was also determined in the sera of patients with TBI only, but it lasted longer (from the 1st until the 7th to 8th week after injury). In the case of patients with TBI and bone fractures a specific pattern of post-traumatic dynamic change of the values of serum bFGF immunoreactivity was observed. Namely, the increase of bFGF immunoreactivity (up to seven-fold above the normal values) was determined even during the first week after injury. Afterwards, periods of high values of bFGF immunoreactivity observed during the 2nd, 4th and the 7-10th weeks after injury were interrupted by sudden decreases even to the normal values (during the 3rd and the 5-6th week after injury).(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of the glomerular filtration rate by identification of sinistrin kinetics.

A computer-based method of system identification and estimation of parameter variance for two-compartment models matched to dynamic sinistrin concentration profiles for the determination of glomerular filtration rate is described. Thereby a procedure for the judgment of the optimal sampling time horizon is presented. Since single-injection techniques are suspected of yielding systematic overestimation of the glomerular filtration rate, a method is demonstrated confirming that such a technique employing sinistrin kinetics can be used to correctly determine the glomerular filtration rate. The validation of the system parameters gained by the single-injection method is made through prediction of the concentration contour under a constant infusion regimen in the same subject on a different occasion. This was performed in healthy controls and in patients with various degrees of renal insufficiency. Upon consideration of the dependence of the clearance estimates and their variances on the protocol duration in test subjects examined from four to ten hours, an adaptive design of the protocol length is developed.

Comparison of the values of basic fibroblast growth factor determined by an immunoassay in the sera of patients with traumatic brain injury and enhanced osteogenesis and the effects of the same sera on the fibroblast growth in vitro.

In patients with severe traumatic brain injury, the early healing of fractures is accompanied by hypertrophic callus formation or heterotopic ossifications, which might even result in ankylosis of the affected joints. Analysis of the sera of patients with traumatic brain injury revealed post-traumatic dynamic changes of basic fibroblast growth factor immunoreactivity, similar to those observed during fracture healing associated with enhanced osteogenesis. The aim of this study was to determine whether such changes in basic fibroblast growth factor concentrations could be related to the phenomenon of enhanced osteogenesis. Basic fibroblast growth factor immunoreactivity was determined (using an IEMA kit) in the sera of patients with traumatic brain injury and bone fractures (n = 8) and in the sera of patients with either traumatic brain injury alone (n = 10) or bone fractures alone (n = 7), and the effects of these sera on L929 fibroblast growth were analysed in vitro. The results did not prove a causative relationship between the changes of basic fibroblast growth factor immunoreactivity and in vitro growth promoting effects of the sera. However, it is apparent that, in addition to changes in the growth-promoting activity and basic fibroblast growth factor concentration of serum, other as yet unknown post-traumatic changes can cause enhanced osteogenesis.

Relationship between generation and plasma concentration of anorganic phosphorus. In vivo studies on dialysis patients and in vitro studies on erythrocytes.

The plasma concentration of inorganic phosphorus (Pi) was determined before, during and after hemodialysis in 28 patients with chronic renal failure. Pi plasma concentration decreased rapidly when hemodialysis was started but did not fall below normal levels during continued dialysis. These changes of Pi concentration were fitted to a model of Pi kinetics in which Pi delivery to plasma is a nonlinear function of the extracellular Pi concentration. In separate in vitro studies, erythrocytes from six subjects with normal renal function and from 14 patients with chronic renal failure were incubated in homologous plasma with various amounts of Pi added. All other factors known to affect the Pi shift between intra - and extracellular fluid compartments (pH, calcium concentration) were kept constant. The relation between Pi concentration in plasma used for incubation and in red cells after 1h incubation suggested a mechanism in which a high plasma concentration results in movement of Pi into red cells where Pi is stored most probably in glucose esters. At low Pi plasma concentration Pi is delivered to plasma at a rate which cannot be explained solely by passive movement of intracellular Pi to plasma but requires additional generation from intracellular storage forms. The generation and delivery of Pi in patients and in their erythrocytes indicate a simple cell-mediated Pi homeostasis counter-acting abnormal fluctuations of plasma Pi.

Post-traumatic dynamic change of carboxyterminal propeptide of type I procollagen, alkaline phosphatase and its isoenzymes as predictors for enhanced osteogenesis in patients with severe head injury

Patients suffering from severe head injury and fractures of long bones or large joints often show enhanced osteogenesis, with hypertrophic callus formation and/or heterotopic ossifications. The advantage of this phenomenon is early consolidation of the fractures. An extreme disadvantage is extensive periarticular calcification, resulting in complete ankylosis of the affected joint. In spite of numerous efforts aimed at clarifying the way in which severe head injury can influence osteogenesis at a distant site, this phenmenon is still not understood. The process, once started seems irreversible, but if diagnosed in time, could be prevented with non-steroid anti-inflammatory drugs that inhibit development of heterotopic ossifications. The major prerequisite for testing this possibility is to define parameters of an early diagnosis of enhanced osteogenesis. Thus, the aim of this study was to test whether serum values of some parameters related to bone regeneration could allow an early prediction of enhanced ossification following bone fracture in patients with severe head injury. Samples of sera were obtained from three groups of injured patients: fractures of long bones or large joints only (n=6), severe head injury only (n=8), severe head injury and fractures of long bones and large joints (n=7) and from a group of apparently healthy volunteers (n=10). The values for alkaline phosphatase (ALP), the bone isoenzyme, and the carboxy terminal propeptide of type I procollagen (PICP) were significantly higher (5–20 times as high) in patients with severe head injury and bone or joint fractures than in any other group. Significantly increased concentrations of PICP were already found in the 1st week after injury, and those of ALP and of the bone isoenzyme increased during the 2nd week after injury. Results show that these parameters are helpful for an early diagnosis of enhanced osteogenesis and heterotopic ossifications in patients with severe head injury and bone fractures. Further studies are necessary to verify these findings, while analysis of reasons for the specific patterns of dynamic change of these parameters could lead to better understanding of the mechanisms underlying the uncontrolled bone formation.

Determination of renal clearance of neopterin by a pharmacokinetic approach

Pharmacokinetic modelling was used to determine the glomerular filtration rate and tubular secretion of neopterin, a marker for cellular immune activation. The method involves parameter identification employing the transient venous plasma concentration profiles of marker substances. By combined i.v. injection of neopterin and inulin which is excreted exclusively via glomerular filtration, neopterin was shown to be excreted in addition to glomerular filtration, by tubular secretion: clearance of inulin, 112 (S.D. 2.2) ml/liter; clearance of neopterin, 499 (S.D. 79.7) ml/min. A pilot experiment using in addition p‐amino hippuric acid suggests that neopterin and p‐amino hippuric acid may employ the same carrier system for tubular secretion.

The Determination of Aluminium in Human Plasma

SummaryDetermination of aluminium in human plasma is of great interest in monitoring dialysis patients under oral aluminium therapy. Flameless atomic-absorption is chosen as the method because of the low normal levels of this non-essential trace element. A method avoiding the analytical problems of aluminium determination in human plasma is described. Normal values for healthy persons and levels for dialysis patients are given.ZusammenfassungDie Bestimmung von Aluminium im menschlichen Plasma ist von großem Interesse bei der Überwachung von Dialysepatienten, die unter oraler Aluminiumtherapie stehen. Die flammenlose Atomabsorption ist die Methode der Wahl, da die Normalwerte dieses nicht essentiellen Spurenelements sehr niedrig sind. Eine Methode, die die analytischen Probleme bei der Bestimmung von Aluminium im menschlichen Plasma vermeidet, wird beschrieben. Normalwerte gesunder Personen und solche von Dialysepatienten werden angegeben.

PMN-related parameters for the monitoring of wound healing in traumatology.

SummaryIn the search for objective methods to monitor the course of wound healing, the proteinase PMN elastase (n=56 pat.), the lipid peroxidation product malondialdehyde (MDA) (n=18 pat.), and polymorphonuclear neutrophil granulocytes (PMN) migratory behaviour were measured [1, 6, 7, 11]. This “stimulated PMN-locomotion” was quantified by a new PMN migration filter assay (n=10 pat.) [2]. We determined the clinical course during “per primam (pp)” wound healing (group 1), “pp” wound healing with secondary inflammatory disease (group 2), manifestation of a bacterial wound infection during healing-“per secundam (ps)” (group 3) and manifest wound infection (“ps”) at the time of admission (group 4).In group 1 PMN elastase returned to normal values on the 10th postsurgical day. Median values in group 3 reflected a highly significant difference (p<0,01) on day 4 and 5 compared with group 1. In group 2 and 4 medians reflected consistent high values without reaching normal ranges throughout. MDA did not exceed the normal range in group 1, in group 3 low levels persisted, and in group 4 a recurring increase was noticed.The total migration index median (TMI) in Group I, which quantifies the percentage of stimulated PMN, reflected its highest value immediately post-surgically and dropped to the lowest on the 13th postsurgical day (decrease by 54%). The mean invasion depth (T/2), a parameter of PMN distribution, showed only slight variation with time. In a group 3-patient, T/2 reflected a maximal migratory stimulation on day 6, 4 days before clinical infection signs could be noticed; then it dropped to the lowest on day 10. This decrease probably reflects a PMN behavioural change from migration to phagocytosis [9].RésuméLa protéinase PMN élastase (n=56 malades), le produit de la peroxidation lipidique malon dialdehyde (MDA) (n=18) aussi bien que la migration des granulocytes neutrophiles polymorphonucléaires (PMN) ont été mesurées dans le cadre de la recherche de méthodes pour surveiller le cours de la cicatrisation. Cette “PMN Locomotion simulée” a été quantifiée par un nouvel essai pour la migration des PMN (n=10). Nous avons déterminé le cours clinique de la cicatrisation dans un groupe de cicatrisation “per primam” (pp) (groupe 1), “pp” cicatrisation et surinfection secondaire (groupe 2), surinfection bactérienne durant la cicatrisation “per secundam” (ps) (groupe 3) et infection initiale au moment de l’admission (groupe 4).Dans le groupe 1 l’élastase PMN est retournée à des valeurs normales au dixième jour après l’intervention. Les valeurs moyennes du groupe 3 ont montré une différence significative (p<0,01) le quatrième et cinquième jour par rapport au groupe 1. Dans les groupes 2 et 4 les moyennes ont montré des valeurs élevées permanentes sans atteindre des valeurs normales. MDA est resté dans les limites de la normale dans le groupe 1; dans le groupe 3 les valeurs sont restées basses et dans le groupe 4 on a pu constater un accroissement répété des valeurs.Dans la migration PMN (concernant le groupe 1), la moyenne de l’index total de migration (TMI), qui mesure le pourcentage de PMN stimulé, présente la valeur la plus élevée immédiatement après l’intervention pour tomber au plus bas le treizième jour après l’intervention (diminution de 54%). La profondeur moyenne d’invasion (T/2), un paramètre de distribution de PMN, a montré seulement une légère variation avec le temps. Avec un malade du groupe 3, T/2 a montré une stimulation migratoire maximale le sixième jour, 4 jours avant que des signes cliniques d’infection aient pu être remarqués; puis T/2 est tombé au plus bas le dixième jour. cette diminution de T/2 reflète probablement des conditions de PMN changeant de la migration à la phagocytose.

Assay of phagocyte activation by means of malondialdehyde and luminol-enhanced chemiluminescence during uneventful wound healing following trauma surgery

The purpose of the study was the assessment of the acute inflammatory response in patients (N = 12) with comparable trauma severity and uneventful wound healing courses in the postsurgical period as a contribution to the search for objectifiable criteria in the monitoring of wound healing. Whole blood chemiluminescence (CL) on the one hand and the lipid peroxidation product malondialdehyde (MDA) on the other hand as tools for the detection of the respiratory burst activity of phagocytes were used as inflammation markers and were compared with the established marker PMN elastase. Blood samples were withdrawn daily from the day of surgery to the 14th postsurgical day. CL-parameters and PMN elastase increased postoperatively reflecting surgical trauma, while MDA remained within the normal range during the whole time of observation. A decrease of CL-activity in the postsurgical period correlated with decreasing PMN elastase levels (r = 0.52, P<0.0001) as well as with the tapering of local inflammation signs concerning the wound situs. MDA values neither correlated with PMN elastase nor with any CL-parameters. The results indicate that the measurement of the phagocytic activation by CL, used for the first time in traumatology to monitor wound healing, represents a promising marker for the assessment of the actual inflammatory status.

System identification of the low-dose kinetics of p-aminohippuric acid.

The renal clearance of p-aminohippuric acid, due to tubular secretion in addition to glomerular filtration, can only be determined by kinetic experiments. Maximal information can be gained from observed temporal marker concentration profiles by fitting dynamic mathematical models of the processes involved, such as absorption, distribution, and elimination, to the kinetic data. Thereby the values of the system constants, such as fractional elimination or fractional distribution rates, and their accuracy measures are determined by methods which are based firstly on measured time-dependent data elicited in an individual test object by perturbing inputs and secondly, on mathematical formulations of prior knowledge of the underlying physiological system. Such methods of model adaptation are called system identification. In this context a computer-based method of system identification and error estimation for the system constants of two-compartment models matched a dynamic concentration profiles of p-aminohippuric acid is presented. The method is used of single-injection experiments to demonstrate that such a technique is able to correctly estimate the clearance of p-aminohippuric acid if sufficiently long experimental protocols are chosen, and to ascertain the sufficient length of a protocol for an individual subject. The renal clearance of p-aminohippuric acid is known to exhibit concentration-dependence generally, but to achieve its maximal value when low doses are applied. The present study deals with the low-dose kinetics of p-aminohippuric acid.