A comparative study of the immune modulating properties of antifibrinolytics in cardiac surgery.

PURPOSE: Antifibrinolytics, used in cardiac surgery to abate postoperative blood loss, share anti-inflammatory properties by suppression of pro-inflammatory D-dimer and plasmin levels. Additional drug specific immune modulating qualities are often mentioned in the discussion on which antifibrinolytic can best be used. To determine the extent and relevance of these effects, we investigated cytokine and growth factor plasma levels in cardiac surgery patients randomized to receive either tranexamic acid, aprotinin, or placebo. Corticosteroid-treated patients served to put the effects in perspective. METHODS: Using a biochip immunoassay, plasma of 36 cardiac surgery patients was quantified for 12 cytokines and growth factors, assessed preoperatively and 6, 12, 24, and 48 h after the start of cardiopulmonary bypass. Eight patients were treated with tranexamic acid, nine with aprotinin, and nine received placebo. Ten placebo-treated patients received corticosteroids. RESULTS: IL-1ß, IL-6, IL-8, IL-10, IFN-γ, TNF-α, VEGF, MCP-1, and EGF plasma concentrations significantly changed over time across all patients. Aprotinin-treated patients showed decreased pro-inflammatory TNF-α and peak MCP-1 plasma levels when compared with placebo. However, corticosteroids attenuated the inflammatory response to a much larger extent, lowering postoperative IL-6, IL-10, IFN-γ, and VEGF concentrations also. CONCLUSIONS: Aprotinin attenuates postoperative pro-inflammatory levels TNF-α and MCP-1 whereas tranexamic acid does not. The majority of plasma proteins studied, however, were not affected by the use of antifibrinolytics when compared with placebo. A clinically relevant common anti-inflammatory effect through inhibition of fibrinolysis seems therefore unlikely.

Arsenic-induced neurotoxicity in relation to toxicokinetics: effects on sciatic nerve proteins.

In our previous study in rats acutely exposed to As, we observed an effect of As on neurofilaments in the sciatic nerve. This study deals with the effects of inorganic As in Wistar rats on the cytoskeletal protein composition of the sciatic nerve after subchronic intoxication. Sodium meta-arsenite (NaAsO2) dissolved in phosphate-buffered saline (PBS) was administered daily in doses of 0, 3 and 10 mg/kg body weight/day (n=9 rats/group) by intragastric route for 4, 8 and 12 week periods. Toxicokinetic measurements revealed a saturation of blood As in the 3- and 10-mg/kg dose groups at approximately 14 microg/ml, with an increase in renal clearance of As at increasing doses. After exsanguination, sciatic nerves were excised and the protein composition was analyzed. Analysis of the sciatic nerves showed compositional changes in their proteins. Protein expression of neurofilament Medium (NF-M) and High (NF-H) was unchanged. Neurofilament protein Low (NF-L) expression was reduced, while mu- and m-calpain protein expression was increased, both in a dose/time pattern. Furthermore, NF-H protein was hypophosphorylated, while NF-L and microtubule-associated protein tau (MAP-tau) proteins were (hyper)-phosphorylated. In conclusion, we show that expression of mu- and m-calpain protein is increased by exposure to As, possibly leading to increased NF-L degradation. In addition, hyperphosphorylation of NF-L and MAP-tau by As also contribute to destabilization and disruption of the cytoskeletal framework, which eventually may lead to axonal degeneration.

Mechanism of arsenic-induced neurotoxicity may be explained through cleavage of p35 to p25 by calpain.

In recent studies we have demonstrated that arsenic (As) metabolites change the composition of neuronal cytoskeletal proteins in vivo and in vitro. To further examine the mechanism of arsenic-induced neurotoxicity with various arsenate metabolites (iAsV, MMAV and DMAV) and arsenite metabolites (iAsIII, MMAIII and DMAIII), we investigated the role of the proteolytic enzyme calpain and its involvement in the cleavage of p35 protein to p25, and also mRNA expression levels of calpain, cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (gsk3ss). A HeLa cell line transfected with a p35 construct (HeLa-p35) was used as a model, since all other proteins such as calpain, CDK5 and GSK3beta are already present in HeLa cells as they are in neuronal cells. HeLa-p35 cells were incubated with various As metabolites and concentrations of 0, 10 and 30 microM for duration of 4 h. Subsequently the cells were either lysed to study their relative quantification levels of these genes or to be examined on their p35-protein expression. P35-RNA expression levels were significantly (p<0.01) increased by arsenite metabolites, while p35 protein was cleaved to p25 (and p10) after incubation with these metabolites. The cleavage of p35 is caused by calcium (Ca2+) induced activation of calpain. Inhibition of calpain activity by calpeptin prevents cleavage of p35 to p25. These results suggest that cleavage of p35 to p25 by calpain, probably As-induced Ca2+-influx, may explain the mechanism by which arsenic induces its neurotoxic effects.

Calcineurin activity and inhibition in skin and (epi)dermal cell cultures.

Calcineurin (Cn) is the target of the immunosuppressive drugs cyclosporine A (CsA), tacrolimus (Trl), and pimecrolimus (Prl). Trl and Prl are often used topically for treatment of various skin diseases. The Cn inhibitors CsA and Trl are mostly used for maintenance therapy of transplant patients. Their long-term use, however, causes a dramatic increase in skin cancer risk. By using a newly developed assay for Cn measurement in blood, we were able to demonstrate Cn activity in total skin homogenates. A significantly higher activity was found in epidermis compared to dermis. In skin cell cultures, fibroblasts showed the highest activity as compared to keratinocytes and melanocytes. Of the Cn inhibitors, Trl showed stronger inhibition than CsA and Prl (57 and 55% in fibroblast and keratinocyte cultures, respectively). Also, the lowest IC(50) (the half maximal inhibitory concentration) values were found for Trl (0.5 and 1.3 nM in two different fibroblast cultures). Cn activity and its inhibition can thus be studied in dermatological samples. The effects of Cn inhibition in fibroblasts and keratinocytes may be of influence on the overall functioning of the skin immune system.

Arsenic metabolites affect expression of the neurofilament and tau genes: an in-vitro study into the mechanism of arsenic neurotoxicity.

Neurological studies indicate that the central (CNS) and peripheral nervous system (PNS) may be affected by arsenic (As). As-exposed patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison to healthy subjects. As may play a role in the disruption of neuroskeletal integrity, but the mechanisms by which it exerts a toxic effect on the peripheral and central nervous system are still unclear. In the present study, we examined the neurotoxic effects of various arsenic metabolites (iAs(III), iAs(V), MMA(V) and DMA(V)) on two different cell lines derived from the peripheral (ST-8814) and central (SK-N-SH) nervous system. The effects of the arsenic metabolites were examined on the relative quantification levels of the cytoskeletal genes, neurofilament-light (NEFL), neurofilament-medium (NEF3), neurofilament-heavy (NEFH) and microtubule-associated protein-tau (MAPT), using real-time PCR. Our results show that iAs(III) and iAs(V) have no significant effects on either cell lines. On the other hand, MMA(V) and DMA(V) cause significant changes in expression levels of NEF3 and NEFL genes, while the expression level of the NEFH gene is significantly increased in both cell lines.

Microparticle-associated tissue factor activity: a link between cancer and thrombosis?

BACKGROUND: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. PATIENT/METHODS: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. RESULTS: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. CONCLUSIONS: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

Arsenic-induced toxicity: effect on protein composition in sciatic nerve.

Exposure to arsenic compounds may lead to skin and lung cancer and various disorders such as vascular disease and peripheral neuropathy in humans. Peripheral arsenic neurotoxicity has been demonstrated clinically and in electrophysiological studies. Patients intoxicated with arsenic show neurological symptoms in their feet and hands. These patients show significantly lower nerve conduction velocities (NCVs) in their peripheral nerves in comparison with controls. The mechanism of arsenic peripheral nervous system (PNS) toxicity, however, has never been described before. This is the first study to investigate the toxicity of arsenic on the PNS. Male Wistar rats were exposed to arsenite given as a single dose i.v. After sacrifice, sciatic nerves were excised and the protein composition was analysed. Protein analysis of sciatic nerves showed disappearance of neurofilament and fibroblast proteins in rats treated with arsenite doses of 15 and 20 mg/kg in comparison with the control groups. Some fibroblast protein bands had disappeared in the 20-mg/kg dose group. The analysed neurofilament-M and -L proteins decreased dose dependency over time. arsenic affects the composition of proteins in the rat sciatic nerve, especially the neurofilaments. The reduction of signals in Western blot analysis reveals changes in cytoskeletal composition, which may well lead to neurotoxic effects in vivo.

Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo.

BACKGROUND: Endothelial activation and dysfunction are associated with several diseases. However, hardly any specific markers are available. Microparticles (MP) from endothelial cells (EC; EMP) were reported in patient groups and healthy individuals. The antibodies used to detect EMP, however, were mainly directed against antigens without EC specificity. OBJECTIVES: We evaluated the antigens on EC and EMP to establish proper markers for EMP detection. METHODS: EMP were isolated from supernatants of resting and interleukin (IL)-1alpha activated human umbilical vein EC (HUVEC; n=3; 0-72 h), stained with annexin V and monoclonal antibodies, and analyzed by flow cytometry. Human platelet-MP (PMP), the main MP population in plasma, were prepared in vitro. EMP and PMP were studied in plasma from systemic lupus erythematosus (SLE) patients (n=11) and healthy individuals (n=10). RESULTS: Platelet-endothelial cell adhesion molecule-1 (PECAM-1), alphanu and beta3 were constitutively exposed on HUVEC, but (almost) absent on EMP (<15% positive for alphanu and beta3), or only exposed on a subpopulation (PECAM-1; 30-60%). Activated HUVEC (>80%) and (subpopulations of) EMP exposed E-selectin and tissue factor. PMP strongly exposed PECAM-1, beta3, and glycoprotein (GP)Ib (CD42b), but not alphanu or E-selectin. GPIb and P-selectin (CD62P) were absent on EMP. Plasma samples contained 0.5% MP staining for E-selectin and/or alphanu. Plasma from one SLE patient contained E-selectin exposing MP (21%), but little alphanu-positive MP. CONCLUSIONS: EC release EMP in vitro. The antigenic phenotype of EMP released from resting and IL-1alpha-stimulated EC differs among each other as well as from resting and stimulated EC, respectively. E-selectin exposed on IL-1alpha-stimulated EC is a valid marker for EMP detection ex vivo to establish endothelial cell activation.

Pro- and non-coagulant forms of non-cell-bound tissue factor in vivo.

BACKGROUND: Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF. METHODS: Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry. RESULTS: In pericardial samples, 45-77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)-mediated thrombin generation in vitro. Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass (P = 0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic. CONCLUSIONS: We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patients.

Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation.

We determined the numbers, cellular origin and thrombin-generating properties of microparticles in healthy individuals (n = 15). Microparticles, isolated from fresh blood samples and identified by flow cytometry, originated from platelets [237 x 10(6)/L (median; range 116-565)], erythrocytes (28 x 10(6)/L; 13-46), granulocytes (46 x 10(6)/L; 16-94) and endothelial cells (64 x 10(6)/L; 16-136). They bound annexin V, indicating surface exposure of phosphatidylserine, and supported coagulation in vitro. Interestingly, coagulation occurred via tissue factor (TF)-independent pathways, because antibodies against TF or factor (F)VII were ineffective. In contrast, in our in vitro experiments coagulation was partially inhibited by antibodies against FXII (12%, p = 0.006), FXI (36%, p <0.001), FIX (28%, p <0.001) or FVIII (32%, p <0.001). Both the number of annexin V-positive microparticles present in plasma and the thrombin-generating capacity inversely correlated to the plasma concentrations of thrombin-antithrombin complex (r = -0.49, p = 0.072 and r = -0.77, p = 0.001, respectively), but did not correlate to prothrombin fragment F1+2 (r = -0.002, p = 0.99). The inverse correlations between the number of microparticles and their thrombin-forming capacity and the levels of thrombin-antithrombin complex in plasma may indicate that microparticles present in the circulation of healthy individuals have an anticoagulant function by promoting the generation of low amounts of thrombin that activate protein C. We conclude that microparticles in blood from healthy individuals support thrombin generation via TF- and FVII-independent pathways, and which may have an anticoagulant function.

Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms.

AIM: We investigated the occurrence and thrombin generating mechanisms of circulating microparticles (MP) in patients with multiple organ dysfunction syndrome (MODS) and sepsis. METHODS: MP, isolated from blood of patients (n = 9) and healthy controls (n = 14), were stained with cell-specific monoclonal antibodies (MoAbs) or anti-tissue factor (anti-TF) MoAb and annexin V, and analyzed by flow cytometry. To assess their thrombin-generating capacity, MP were reconstituted in normal plasma. The coagulation activation status in vivo was quantified by plasma prothrombin fragment F1+2- and thrombin-antithrombin (TAT) measurements. RESULTS: Annexin V-positive MP in the patients originated predominantly from platelets (PMP), and to a lesser extent from erythrocytes, endothelial cells (EMP) and granulocytes (GMP). Compared to healthy controls, the numbers of annexin V-positive PMP and TF-exposing MP were decreased (p = <0.001 for both), EMP were decreased (E-selectin, p = 0.003) or found equal (CD144, p = 0.063), erythrocyte-derived MP were equal (p = 0.726), and GMP were increased (p = 0.008). GMP numbers correlated with plasma concentrations of elastase (r = 0.70, p = 0.036), but not with C-reactive-protein or interleukin-6 concentrations. Patient samples also contained reduced numbers of annexin V-negative PMP, and increased numbers of erythrocyte-derived MP and GMP (p = 0.005, p = 0.021 and p <0.001, respectively). Patient MP triggered thrombin formation, which was reduced compared to the healthy controls (p = 0.008) and strongly inhibited by an anti-factor XII MoAb (two patients), by anti-factor XI MoAb (eight patients) or by anti-TF MoAb (four patients). Concentrations of F1+2 and TAT were elevated (p = 0.005 and p = 0.001, respectively) and correlated inversely with the number of circulating MP (and r = -0.51, p = 0.013, and r = -0.65, p = 0.001, respectively) and their thrombin generation capacity (F1+2: r= -0.62, p = 0.013). CONCLUSIONS: In patients with MODS and sepsis relatively low numbers of MP are present that differ from controls in their cellular origin, numbers and coagulation activation mechanisms.

Dexamethasone concentration in the subretinal fluid after a subconjunctival injection, a peribulbar injection, or an oral dose.

PURPOSE: To determine dexamethasone concentrations in the subretinal fluid of patients after a peribulbar injection, a subconjunctival injection, or an oral dose of dexamethasone and to compare the results with those of previous similar studies of dexamethasone concentrations in the vitreous. DESIGN: Prospective, nonrandomized, comparative trial. PARTICIPANTS: One hundred forty-eight patients with a rhegmatogenous retinal detachment. METHODS: Fifty patients received a peribulbar injection of 5 mg dexamethasone disodium phosphate, 49 received a subconjunctival injection of 2.5 mg dexamethasone disodium phosphate, and 49 received an oral dose of 7. 5 mg dexamethasone at various time intervals before surgery. At the time of surgery, a subretinal fluid sample was taken from each patient. MAIN OUTCOME MEASURES: The dexamethasone concentration in the subretinal fluid measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentrations in the subretinal fluid after the peribulbar injection, the subconjunctival injection, and the oral dose were, respectively, 82.2 ng/ml (standard error, 17. 6), 359 ng/ml (standard error, 80.2), and 12.3 ng/ml (standard error, 1.61). Corrected for dose, the maximum dexamethasone concentrations after subconjunctival injection and peribulbar injection were, respectively, 120 (95% confidence interval, 54/180) and 13 (95% confidence interval, 6.8/20) times greater than after oral administration. CONCLUSIONS: A subconjunctival injection of dexamethasone disodium phosphate is more effective in delivering dexamethasone into the subretinal fluid of patients with a rhegmatogenous retinal detachment compared with peribulbar injection or oral administration. The subretinal dexamethasone concentrations were higher than concentrations measured in the vitreous in previous studies with a similar setup after all three delivery methods.

Activated platelets in patients with severe hypertriglyceridemia: effects of triglyceride-lowering therapy.

Hypertriglyceridemia, a risk factor for cardiovascular disease, has been associated with hypercoagulability, but whether platelet activation is implicated is unknown. This study was designed to compare the in vivo platelet activation status between patients with severe hypertriglyceridemia and age- and sex-matched control subjects, and to evaluate the effects of triglyceride-lowering therapy. Sixteen patients with primary hypertriglyceridemia were included in a double-blind, placebo-controlled cross-over trial with 400 mg bezafibrate once daily. Platelet activation was analysed by double label flow cytometry, using monoclonal antibodies against GP53, P-selectin, and platelet-bound fibrinogen. Surface expression of the lysosomal membrane protein GP53 was significantly higher in the hypertriglyceridemic patients at baseline as compared to the group of age- and sex-matched controls (16.3+/-4.8% vs. 8.9+/-3.4%, respectively, P<0.001). No differences in the expression of P-selectin and fibrinogen binding were observed. In response to bezafibrate therapy, the expression of GP53 in the patient group decreased from 16.3+/-4.8% to 13.1+/-4.1% (P=0.018). The expression of P-selectin and fibrinogen binding was not affected by bezafibrate therapy. In conclusion, patients with hypertriglyceridemia have an increased in vivo platelet activation status, which can be improved by bezafibrate therapy.

The amount of free corticosterone is increased during lipopolysaccharide-induced fever.

The relation between lipopolysaccharide (LPS)-induced fever and bioavailability of corticosterone (B) was examined in male Wistar rats. Animals were injected with LPS (2.5 mg/kg i.p.) or saline and core temperature and heart rate were monitored continuously using a biotelemetry system. Blood samples were withdrawn from freely moving rats via jugular catheters for estimation of total and free plasma B. LPS induced a long-lasting increase (24-48 h) in core temperature and B secretion and a short-lasting increase (90 min) in heart rate. LPS-induced fever was accompanied by a significant increase in the free/total B ratio. In contrast, an acute injection of B, which resulted in circulating B levels similar to those found after LPS, did not affect the free/total B ratio. The important role of LPS-induced fever in the hormone secretion pattern and the equilibrium between free and total B was further demonstrated in an in vitro study showing that an increase in the temperature by 3 degrees C elevated the free B fraction and the free/total B ratio of plasma samples with concentrations of B in the physiological range (5-40 microg/dl). Taken together, these findings indicate that during LPS-induced fever there is an increase in the amount of biologically available B. Exposure of glucocorticoid-sensitive targets to elevated levels of free B could contribute to the restoration of homeostasis that is disturbed during inflammation.

Cellular origin and procoagulant properties of microparticles in meningococcal sepsis.

Patients with meningococcal sepsis generally suffer from disseminated intravascular coagulation (DIC). The aim of this study was to address whether these patients have elevated numbers of circulating microparticles that contribute to the development of DIC. Plasma samples from 5 survivors, 2 nonsurvivors, and 5 healthy volunteers were analyzed for the presence of microparticles by flow cytometry. Ongoing coagulation activation in vivo was quantified by enzyme-linked immunosorbent assay of plasma prothrombin fragment F(1 + 2), and procoagulant properties of microparticles in vitro were estimated by thrombin-generation assay. On admission, all patients had increased numbers of microparticles originating from platelets or granulocytes when compared with controls (P =.004 and P =.008, respectively). Patients had elevated levels of F(1 + 2) (P =.004), and their microparticles supported thrombin generation more strongly in vitro (P =.003) than those of controls. Plasma from the patient with the most fulminant disease course and severe DIC contained microparticles that expressed both CD14 and tissue factor, and these microparticles demonstrated extreme thrombin generation in vitro. We conclude that patients with meningococcal sepsis have elevated numbers of circulating microparticles that are procoagulant. These findings may suggest a novel therapeutic approach to combat clinical conditions with excessive coagulation activation.

High concentration of dexamethasone in aqueous and vitreous after subconjunctival injection.

PURPOSE: To determine the dexamethasone concentration in aqueous, vitreous, and serum of patients after a subconjunctival injection with dexamethasone disodium phosphate and to compare the effectiveness of a subconjunctival injection as a method of delivering dexamethasone into the vitreous with that of two previously tested routes: peribulbar injection and oral administration. METHODS: In a prospective study, 50 phakic patients who underwent a pars plana vitrectomy received a single subconjunctival injection with 2.5 mg of dexamethasone disodium phosphate, aqueous solution (after topical anesthesia and a subconjunctival injection with lidocaine) at varied intervals before surgery. An aqueous and a vitreous sample were taken from each patient, and serum samples were collected at multiple time points from nine of 50 patients. Dexamethasone concentrations were measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentration in the aqueous was 858 ng per ml at 2.5 hours after injection, and in the vitreous, 72.5 ng per ml at 3 hours. In serum, a mean maximum concentration of 32.4 ng per ml was measured at approximately 30 minutes after injection. CONCLUSIONS: Subconjunctival injection of 2.5 mg of dexamethasone disodium phosphate resulted in an estimated vitreous dexamethasone peak concentration three and 12 times higher, respectively, than after a peribulbar injection of 5 mg of dexamethasone disodium phosphate and an oral dose of 7.5 mg of dexamethasone. Thus, a subconjunctival injection is the most effective method of delivering dexamethasone into both the anterior and posterior segments of the eye. Systemic drug absorption is considerable and is of the same order of magnitude as after peribulbar injection.

Temperature-dependent cortisol distribution among the blood compartments in man.

This study was conducted to investigate the effect of temperature on the amount of cortisol bound to the erythrocytes and the distribution of cortisol in whole blood at various temperatures. The amount of cortisol bound to the erythrocytes was determined in a way that did not disturb the equilibrium distribution of cortisol between plasma and erythrocytes. Total and free cortisol concentrations in plasma and the amount of cortisol bound to the erythrocytes were determined at 20, 30, 37, and 40 C in the blood of six healthy persons. The amount of cortisol bound to the erythrocytes showed a perfect linear relation with the free cortisol concentration and was independent from the temperature. The average ratio of the erythrocyte-associated and free cortisol was 2.38 +/- 0.06. Computer simulations of the distribution of cortisol among the blood compartments showed that the free and loosely bound fraction (albumin and erythrocytes) was highly temperature dependent: at 30 C, this fraction was 3-5 times lower than at 37 C. It was demonstrated by computer simulation that changes in the concentration of cortisol-binding globulin had an effect on the fractional distribution of cortisol among the blood components. These shifts in the cortisol distribution, between the erythrocyte and the plasma compartment, can also be the cause of apparently high or low free and total plasma cortisol concentrations. Differences up to 25% in the free cortisol concentration can be observed. We conclude that the erythrocyte-associated cortisol fraction is relatively undervalued but can serve as an important transport vehiculum and storage compartment for cortisol. This fraction can have a considerable effect on the total plasma and free cortisol concentration when strict temperature control during sample handling is not considered.