Maria Klaude

Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis

Patients with sepsis in the ICU (intensive care unit) are characterized by skeletal muscle wasting. This leads to muscle dysfunction that also influences the respiratory capacity, resulting in prolonged mechanical ventilation. Catabolic conditions are associated with a general activation of the ubiquitin-proteasome pathway in skeletal muscle. The aim of the present study was to measure the proteasome proteolytic activity in both respiratory and leg muscles from ICU patients with sepsis and, in addition, to assess the variation of proteasome activity between individuals and between duplicate leg muscle biopsy specimens. When compared with a control group (n=10), patients with sepsis (n=10) had a 30% (P<0.05) and 45% (P<0.05) higher proteasome activity in the respiratory and leg muscles respectively. In a second experiment, ICU patients with sepsis (n=17) had a 55% (P<0.01) higher proteasome activity in the leg muscle compared with a control group (n=10). The inter-individual scatter of proteasome activity was larger between the patients with sepsis than the controls. We also observed a substantial intra-individual difference in activity between duplicate biopsies in several of the subjects. In conclusion, the proteolytic activity of the proteasome was higher in skeletal muscle from patients with sepsis and multiple organ failure compared with healthy controls. It was shown for the first time that respiratory and leg muscles were affected similarly. Furthermore, the variation in proteasome activity between individuals was more pronounced in the ICU patients for both muscle types, whereas the intra-individual variation between biopsies was similar for ICU patients and controls.

Protein metabolism and gene expression in skeletal muscle of critically ill patients with sepsis

Muscle wasting negatively affects morbidity and mortality in critically ill patients. This progressive wasting is accompanied by, in general, a normal muscle PS (protein synthesis) rate. In the present study, we investigated whether muscle protein degradation is increased in critically ill patients with sepsis and which proteolytic enzyme systems are involved in this degradation. Eight patients and seven healthy volunteers were studied. In vivo muscle protein kinetics was measured using arteriovenous balance techniques with stable isotope tracers. The activities of the major proteolytic enzyme systems were analysed in combination with mRNA expression of genes related to these proteolytic systems. Results show that critically ill patients with sepsis have a variable but normal muscle PS rate, whereas protein degradation rates are dramatically increased (up to 160%). Of the major proteolytic enzyme systems both the proteasome and the lysosomal systems had higher activities in the patients, whereas calpain and caspase activities were not changed. Gene expression of several genes related to the proteasome system was increased in the patients. mRNA levels of the two main lysosomal enzymes (cathepsin B and L) were not changed but, conversely, genes related to calpain and caspase had a higher expression in the muscles of the patients. In conclusion, the dramatic muscle wasting seen in critically ill patients with sepsis is due to increased protein degradation. This is facilitated by increased activities of both the proteasome and lysosomal proteolytic systems.

Autophagic-lysosomal pathway is the main proteolytic system modified in the skeletal muscle of esophageal cancer patients

BACKGROUND In cancer cachexia, muscle depletion is related to morbidity and mortality. Muscle-wasting mechanisms in cancer patients are not fully understood. OBJECTIVE We investigated the involvement of the proteolytic systems (proteasome, autophagic-lysosomal, calpain, and caspase) in muscle wasting during cancer cachexia. DESIGN Esophageal cancer patients [n = 14; mean ± SD age: 64.1 ± 6.6 y] and weight-stable control patients undergoing reflux surgery (n = 8; age: 57.5 ± 5.8 y) were included. Enzymatic activities were measured in the vastus lateralis and diaphragm. Protein expressions were also measured in the vastus lateralis of control (n = 7) and cancer (n = 8) patients. RESULTS Proteasome, calpain, and caspase 3 activities in the vastus lateralis and diaphragm muscles did not differ between the 2 groups. Cathepsin B and L activities were 90% (± SD) [2.4 ± 0.2 compared with 1.3 ± 0.2 pmol 7-amido-4-methylcoumarin (AMC) · μg protein⁻¹ · min⁻¹; P < 0.001] and 115% (5.3 ± 0.4 compared with 2.5 ± 0.3 pmol AMC · μg protein⁻¹ · min⁻¹; P < 0.001) greater, respectively, in the vastus lateralis of cancer patients than in that of control subjects. We observed (in conjunction with increased lysosomal protease activities) higher microtubule-associated protein 1 light chain 3B-II/I ratios (0.14 ± 0.08 compared with 0.04 ± 0.04) and cathepsin B and L expressions in the vastus lateralis of cancer patients than in that of control subjects (P < 0.05). Protein expression of p62 in the vastus lateralis did not differ between the 2 groups. CONCLUSIONS The autophagic-lysosomal pathway in the skeletal muscle of cancer patients was modified, whereas other proteolytic systems were unchanged. These findings suggest involvement of the autophagic-lysosomal proteolytic system during cancer cachexia development in humans.

Whole body protein turnover in critically ill patients with multiple organ failure

BACKGROUND & AIMS To evaluate the effect of nutrition therapy on protein turnover in critically ill patients isotopically labeled amino acids can be used. Here parallel measurements using (13)C-leucine and (2)H5-phenylalanine were performed to evaluate if one tracer was to be preferred. METHODS As a reference group, healthy volunteers (n = 8) were studied in the postabsorptive state and during parenteral nutrition delivery. ICU patients with multiple organ failure (n = 8) were studied during parenteral nutrition delivery only. RESULTS For the volunteers, the net protein balances changed from negative to positive during parenteral nutrition delivery (compared to the postabsorptive state) when evaluated with leucine and phenylalanine (P < 0.0001). For phenylalanine this change was attributable to an increased protein synthesis (P < 0.0001), while for leucine the change was attributable to a decreased protein degradation (P < 0.0001). For the patients, only measured during parenteral nutrition delivery, the estimates by the two amino acid tracers agreed, showing a protein balance not statistically significantly different from zero. The whole body protein turnover was higher than that of the healthy volunteers during parenteral nutrition delivery. In the patients, the net protein balance correlated positively to the amount of amino acids given. CONCLUSIONS Critically ill patients with multiple organ failure have an increased protein turnover. The findings in the healthy volunteers indicate that the use of the two different amino acid tracers in parallel in future studies should be considered.

Synthesis rates of total liver protein and albumin are both increased in patients with an acute inflammatory response

The general perception that catabolism and inflammation are associated with a high synthesis rate of total liver protein and a low albumin synthesis rate has been challenged in recent years by several studies in man, indicating that the synthesis rate of albumin in response to a catabolic insult is increased rather than decreased. Thus changes in liver protein synthesis rates in conjunction with catabolism and acute inflammation in man need to be characterized better. The aim of the present study was to measure protein synthesis rates of total liver protein and albumin during a state of acute inflammation. Patients (n = 10) undergoing acute laparoscopic cholecystectomy due to acute cholecystitis were investigated. FSRs (fractional synthesis rates) of total liver protein (liver biopsy specimens) and albumin (plasma samples) were investigated as early as possible during the surgical procedure, using a flooding dose of L-[2H5]phenylalanine. The results were compared with a reference group of patients without cholecystitis undergoing elective laparoscopic cholecystectomy (n = 17). FSR of total liver protein was 60% higher (P < 0.001) and the FSR of albumin was 45% higher (P < 0.01) in the cholecystitis patients compared with the control group. In conclusion, the synthesis rates of total liver protein and albumin are both increased in patients with an acute general inflammatory reaction undergoing laparoscopic cholecystectomy.

Lactate kinetics and mitochondrial respiration in skeletal muscle of healthy humans under influence of adrenaline

Plasma lactate is widely used as a biomarker in critical illness. The aims of the present study were to elucidate the usefulness of a three-compartment model for muscle lactate kinetics in humans and to characterize the response to an exogenous adrenaline challenge. Repeated blood samples from artery and femoral vein together with blood flow measurements and muscle biopsies were obtained from healthy male volunteers (n=8) at baseline and during an adrenaline infusion. Concentrations of lactate and enrichment of [13C]lactate were measured and kinetics calculated. Mitochondrial activity, glycogen concentration, oxygen uptake and CO2 release were assessed. The adrenaline challenge increased plasma lactate 4-fold as a result of a greater increase in the rate of appearance (R(a)) than the increase in the rate of disappearance (R(d)). Leg muscle net release of lactate increased 3.5-fold, whereas intramuscular production had a high variation but did not change. Mitochondrial state 3 respiration increased by 30%. Glycogen concentration, oxygen uptake and CO2 production remained unchanged. In conclusion a three-compartment model gives additional information to the two-compartment model but, due to its larger variation and invasive muscle biopsy, it is less likely to become a regularly used tool in clinical research. Hyperlactataemia in response to adrenergic stimuli was driven by an elevated lactate release from skeletal muscle most probably due to a redirection of a high intramuscular turnover rather than an increased production.

Determination of in vivo protein synthesis in human palatine tonsil

The palatine tonsils are constantly exposed to ingested or inhaled antigens which, in turn, lead to a permanent activation of tonsillar immune cells, even in a basic physiological state. The aim of the present study was to investigate if the immunological activation of the human palatine tonsil is reflected by a high metabolic activity, as determined by in vivo measurement of protein synthesis. The protein synthesis rate of the tonsil was also compared with that of the circulating T-lymphocytes, the total blood mononuclear cells and the whole population of blood leucocytes. Phenotypic characterization of immune-competent cells in tonsil tissue and blood was performed by flow cytometry. Pinch tonsil biopsies were taken after induction of anaesthesia in healthy adult patients (n=12) scheduled for ear surgery, uvulopalatopharyngoplasty or nose surgery. Protein synthesis was quantitatively determined during a 90-min period by a flooding-dose technique. The in vivo protein synthesis rate in the palatine tonsils was 22.8+/-5.7%/24 h (mean+/-S.D.), whereas protein synthesis in the circulating T-lymphocytes was 10.7+/-3.4%/24 h, in mononuclear cells was 10.8+/-2.8%/24 h and in leucocytes was 3.2+/-1.2%/24 h. CD3+ lymphocytes were the most abundant cell population in the tonsil. The in vivo protein synthesis rate in human tonsils was higher compared with the circulating immune cells. This high metabolic rate may reflect the permanent immunological activity present in human tonsils, although cell phenotypes and activity markers do not explain the differences.

A Tracer Bolus Method for Investigating Glutamine Kinetics in Humans

Glutamine transport between tissues is important for the outcome of critically ill patients. Investigation of glutamine kinetics is, therefore, necessary to understand glutamine metabolism in these patients in order to improve future intervention studies. Endogenous glutamine production can be measured by continuous infusion of a glutamine tracer, which necessitates a minimum measurement time period. In order to reduce this problem, we used and validated a tracer bolus injection method. Furthermore, this method was used to measure the glutamine production in healthy volunteers in the post-absorptive state, with extra alanine and with glutamine supplementation and parenteral nutrition. Healthy volunteers received a bolus injection of [1-13C] glutamine, and blood was collected from the radial artery to measure tracer enrichment over 90 minutes. Endogenous rate of appearance (endoRa) of glutamine was calculated from the enrichment decay curve and corrected for the extra glutamine supplementation. The glutamine endoRa of healthy volunteers was 6.1±0.9 µmol/kg/min in the post-absorptive state, 6.9±1.0 µmol/kg/min with extra alanyl-glutamine (p = 0.29 versus control), 6.1±0.4 µmol/kg/min with extra alanine only (p = 0.32 versus control), and 7.5±0.9 µmol/kg/min with extra alanyl-glutamine and parenteral nutrition (p = 0.049 versus control). In conclusion, a tracer bolus injection method to measure glutamine endoRa showed good reproducibility and small variation at baseline as well as during parenteral nutrition. Additionally, we showed that parenteral nutrition including alanyl-glutamine increased glutamine endoRa in healthy volunteers, which was not attributable to the alanine part of the dipeptide.

Repeated quantitative measurements of De Novo synthesis of albumin and fibrinogen

The possibility of using two different isotopomers, for the incorporation of isotopically labeled amino acids, was explored to enable longitudinal studies of de novo synthesis of two export liver proteins, albumin and fibrinogen. The agreement of the synthesis rates between the two different labels was evaluated along with the reproducibility of repeated experiments using different time intervals. Healthy volunteers were studied in a standardized fed state. Protocol A (n = 10) involved two measurements 48 hours apart. Protocol B (n = 6) involved three measurements at baseline and five hours and then seven days after the initial measurement. De novo synthesis of albumin and fibrinogen by the incorporation of D5-phenylalanine or D8-phenylalanine were measured using the flooding dose technique. Albumin and fibrinogen were isolated from plasma using standard techniques. Fractional and absolute synthesis rates were calculated. Repeated measurements employing the two isotoptomers showed good agreement for albumin fractional synthesis rate after 48 hours (p = 0.92) and after 7 days (p = 0.99), with a coefficient of variation of 5.9% when using the same isotopic label. For fibrinogen, the coefficient of variation for the fractional synthesis rate employing the same isotopic label was 16.6%. Repeated measurements after 48 hours and seven days showed less agreement although there was no statistical difference (P = 0.32 and P = 0.30 respectively). Repeated measurement after five hours showed a statistical significant difference for the fractional synthesis rate of fibrinogen (p = 0.008) but not for albumin (p = 0.12). Repeated measurements of albumin de novo synthesis more than 48 hours apart show acceptable agreement using either one or two different isotopic labels. For fibrinogen the larger intra-individual scatter necessitates larger study groups to detect changes in longitudinal studies. Repeated measurements within 48 hours need to be validated further.

Enhanced in vivo protein synthesis in circulating immune cells of ICU patients.

Insufficient function of the immune system contributes to a poor prognosis in intensive care unit (ICU) patients. However, the immune system function is not easily monitored and evaluated. In vivo protein synthesis determination in immune competent cells offers a possibility to quantify immunological activation. The aim of this descriptive study was to determine the in vivo fractional protein synthesis rate (FSR) in immune cells of ICU patients during the initial phase of the critical illness. Patients (n = 20) on ventilator treatment in the general ICU were studied during their first week of ICU stay. FSR was determined in circulating T lymphocytes, mononuclear cells, the whole population of blood leukocytes, and in stationary immune cells of palatine tonsils during a 90-min period by a flooding technique. Healthy, adult subjects (n = 11), scheduled for elective ear, nose, and throat surgery served as a control group. The FSR in leukocytes and mononuclear cells of ICU patients was higher compared with the control group. In contrast, the FSR of circulating T lymphocytes and of tonsillar cells was not different from that in the healthy subjects. In summary, the ICU patients showed a distinct polarization of metabolic responses during the initial phase of the critical illness. The in vivo rate of protein synthesis was high in the circulating mononuclear cells and leukocytes, reflecting enhanced metabolic activity in these cell populations. Determination of the in vivo protein synthesis rate may be used as a tool to obtain additional information on activation of the immune system.