A spectroscopic and molecular dynamics study on the aggregation process of a long-acting lipidated therapeutic peptide: the case of semaglutide.

The aggregation properties of semaglutide, a lipidated peptide drug agonist of the Glucagon-like peptide 1 receptor recently approved for the treatment of type 2 diabetes, have been investigated by spectroscopic techniques (UV-Vis absorption, steady-state and time-resolved fluorescence, and electronic circular dichroism) and molecular dynamics simulations. We show that in the micromolar concentration region, in aqueous solution, semaglutide is present as monomeric and dimeric species, with a characteristic monomer-to-dimer transition occurring at around 20 µM. The lipid chain stabilizes a globular morphology of the monomer and dimer species, giving rise to a locally well-defined polar outer surface where the lipid and peptide portions are packed to each other. At very long times, these peptide clusters nucleate the growth of larger aggregates characterized by blue luminescence and a ß-sheet arrangement of the peptide chains. The understanding of the oligomerization and aggregation potential of peptide candidates is key for the development of long acting and stable drugs.

Effects of recombinant human activated protein C on the fibrinolytic system of patients undergoing conventional or tight glycemic control.

AIM: Recombinant human activated protein C (rh-APC) and tight glycemic control (TGC) have been shown to reduce mortality in septic patients. Both interventions can reduce the plasma concentration and/or activity of the most powerful suppressor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1). Our aim was to evaluate the effects on the fibrinolytic system after the administration of rh-APC in septic patients undergoing conventional or TGC. METHODS: Posthoc analysis of data was collected from 90 patients with severe sepsis/septic shock, randomized to either conventional or TGC groups. Independent of these treatments, patients with at least two organ dysfunctions simultaneously received rh-APC. Plasma levels of multiple biochemical markers for fibrinolysis, coagulation, and inflammation were determined every day for the 1st week and then on study days 9, 11, 13, 18, 23, and 28. Clinical data and sepsis-related organ failure assessment (SOFA) scores were also recorded. RESULTS: Patients who had received rh-APC exhibited significantly more impairments in fibrinolysis at baseline (PAI-1 activity 49.76 [24.61-71.82] vs 21.92 [6.47-55-83] IU/mL, P=0.03). The reductions in plasma PAI-1 activity over time associated with rh-APC treatment were different according to whether the treatment was administered to patients undergoing conventional or TGC (P=0.01). However, the most prominent reductions were in patients undergoing conventional glycemic control. Significant interactions between the two study interventions were also found for PAI-1 concentration (P<0.001), C-reactive protein (P=0.02), and interleukin-6 levels (P<0.001). CONCLUSIONS: Both rh-APC and TGC appear to improve fibrinolysis in septic patients. The reduction in the impairment of fibrinolysis associated with rh-APC treatment seems greater in patients undergoing conventional glycemic control than in those undergoing TGC.

Heat stress: characteristics, pathophysiology and avoidable mistakes.

In August 2003 an exceptional heatwave was recorded in Europe. The authors would like to describe 6 patients for which the intensivist was called as a consultant. All patients had a skin temperature >40 degrees C, central nervous system impairment, severe hyponatremia [124.7 mEq/l+/-5.6 (range 117-130)] and severe metabolic acidosis [BE -6.28 mEq/l+/-3.55 (range -9.5-0), HCO3- 17.75 mEq/l+/-3.25 (range 13.4-21.9)]. All patients had decreased platelet count and coagulation abnormalities. Two patients were hypertensive, 4 hypotensive. The heat stress due to the hot environment is characterized by systemic inflammatory response (as in severe sepsis) and hemodynamic impairment (as in hypovolemic shock). The association between hypovolemia and altered microcirculation leads to cell energy failure with metabolic lactic acidosis. The energy failure may induce structural irreversible damage of mitochondria. It is possible to differentiate, during energy failure, the irreversible or reversible condition by volume loading and vasoactive drugs challenge tests. In fact, if the hemodynamic correction is associated with normalization of SvO2 with disappearance of metabolic acidosis, this suggests hemodynamic impairment with intact mitochondrial function. In contrast, if the hemodynamic improvement with normalization of SvO2 is associated and acidosis persists, this suggests irreversible structural mitochondrial damage. The threshold between reversibility and irreversibility is likely time dependent, as suggested by biochemical consideration and by 2 large randomized studies on hemodynamic treatment. The comparative analysis of these 2 studies suggests that the time of intervention may lead to significant differences in mortality. In these patients time is essential.

A blunt complex abdominal trauma: total hepatectomy and liver transplantation.

A victim of a motor accident was referred to the Emergency Department. He was unconscious, breathing was rapid and shallow, and had a mean arterial pressure of 60 mmHg with a distended abdomen. A peritoneal lavage was positive for blood. An immediate laparotomy showed a massive hepatic injury with bilobar disruptions. After an unsuccessful hepatorrhaphy an extensive perhepatic gauze packing was done. During the operation the patient was massively transfused and high doses of dopamine were used. At the end a relative stability was reached, but a few hours later, due to the high risk of sepsis and abdominal compartment syndrome, a second laparotomy was performed. Despite efforts to reach a good hemostasis, it was decided to perform a total hepatectomy and portocaval shunt. The patient was put on the waiting list for a compatible liver. After 36 h in this anhepatic state, the patient received the new graft. The graft showed immediate recovery. In cases of severe and complex liver trauma, performing a total hepatectomy followed by liver transplantation is justified.

[Neurologic prognosis after cardiocirculatory arrest outside the hospital]. / Prognosi neurologica dopo arresto cardiocircolatorio extraospedaliero.

OBJECTIVE: To detect clinical signs and times of evaluation able to identify patients with good neurologic prognosis after out-of-hospital cardiac arrest. EXPERIMENTAL DESIGN: Retrospective study with 6 months-2 years of follow-up. SETTING: General intensive care unit, from 1993 to 1995. PATIENTS: Twenty-three patients aged 18-80 years, admitted with coma after out-of-hospital cardiac arrest, who survived at least 24 hours, without concomitant neurologic disorders. INTERVENTIONS: Neurologic outcome classification in three categories: complete neurologic recovery (A), moderate to severe disability (B), death without regained consciousness (C); A and B groups of patients are considered together as a "good neurologic outcome" group. MEASUREMENTS: Evaluation of some neurological parameters (GCS, motor response to pain, spontaneous motility, cranial nerve reflexes, spontaneous breathing, seizures) 6 hours (t0), 24 hours (t1), 72 hours (t2) and 7 days (t3) after arrest. RESULTS: Patients with good prognosis are identified with 100% sensitivity from the following parameters: at t0 none; at t1 motor response to pain, GCS > 4, absence of seizures; at t2 the former parameters, spontaneous breathing, brain stem reflexes, normal pupils; at t3 all the former parameters and spontaneous motility. Specificity is greater than 50% only for motor response to pain and GCS > 4. CONCLUSIONS: It is mostly impossible to state a neurologic prognosis in the first hours after cardiac arrest. The single predictor with the most reliable prognostic value is motor response to pain which allows, if present, to identify all the patients with a good prognosis as early as 24 hours after arrest.

Early metabolic treatment after liver transplant: amino acid tolerance.

OBJECTIVE: We investigated the amino acid (AA) tolerance during Total Parenteral Nutrition (TPN) in adult patients undergone liver transplant (LTX). DESIGN: The treatment (Glucose and AA), induced on the 2nd postoperative day, was later maintained with 27 kcal/kg Ideal Body Weight (IBW) as glucose and 0.12 (12 patients: protocol #1), 0.18 (10 patients: protocol #2) and 0.25 g nitrogen (N)/kg IBW (13 patients: protocol #3) till end of the 6th postoperative day. The N intake was sequentially modified in protocol #2 and #3 to increase the supply of the amino acid (AA) that resulted in an infusion plasma level below the expected "normal" range (between 1 and 1.6 times the overnight fasting plasma level of volunteer). PATIENTS: 35 consecutive adult patients without diabetes and organ failures for the entire study period. MEASUREMENTS: Plasma AA profile was measured before LTX and at the last TPN day under continuous infusion. During #1 and #2 protocol, many AA resulted below or at the lower range of the norm while, during 0.25 gN/kg IBW infusion, the majority of the administered AA significantly increased with respect to reference values. Nevertheless, they remained in the "normal" plasma range indicating that they were supplied in an optimal amount (particularly the aromatic and sulphurated ones, potentially toxic if liver function is impaired, and the branched chain AA (BCAA) given at consistent dosage: 0.5 g/kg). Arginine resulted significantly increased (Arg: 1.9 times the reference) and cystine (Cys: 0.45), serine (Ser: 0.8) and taurine (Tau: 0.85) remained significantly lower than "normal" as well as the not administered citrulline (Cit: 0.58) and alfa amino butyric acid (Aba: 0.41). The AA (and calorie) load almost balanced the N losses during the 5th (0.411 +/- 0.038) and 6th study day (0.305 +/- 0.019 gN/kg). CONCLUSIONS: 0.25 gN/kg could be considered the minimum N load in the uncomplicated adult LTX recipients, for reassuring a balanced plasma AA pattern and body N turnover in the early postoperative phase.

Kinetic of body nitrogen loss during a whole day infusion and withdrawal of glucose and insulin in injured patients.

OBJECTIVE: To investigate the kinetics of body nitrogen (N) excretion during 24 h glucose infusion (relating glycemia with insulin supply) and during subsequent 24 h saline infusion in injured patients during a full blown stress reaction. To define the lag time between the start of the withdrawal of glucose and insulin infusion, and the modification in the N loss from the body, and the time span to reach the maximum effect and its size. The knowledge of these variables is mandatory to plan short term studies in critically ill patients, while assuring the stability of the metabolic condition during the study period, and also to assess the possible weaning of the effect on protein breakdown during prolonged glucose and insulin infusion. DESIGN: 24-36 h after injury, patients were fasted ( < 100 g glucose) for 24 h (basal day). Thereafter, a 24 h glucose infusion in amount corresponding to measured fasting energy production rate (EPR), clamping glycemia at normal level with insulin supply followed by 24 h saline infusion, was performed. Total N, urea and 3-methyl-histidine (3-MH) in urine were measures on 4 h samples starting from 20th h of the basal day. SETTING: Multipurpose ICU in University Hospital. PATIENTS: 6 consecutive patients who underwent accidental and/or surgical injury, immediately admitted for respiratory assistance (FIO2 < 0.04). Excluded patients were those with abnormal nutritional status, cardiovascular compromise and organ failures. MAIN RESULTS: Patients showed a 33% increase in measured versus predicted fasting EPR and a consistent increase in N and 3-MH urinary loss. An infusion of glucose at 5.95 +/- 0.53 mg/kg x min (97.20 +/- 0.03% of the fasting measured EPR) with 1.22 +/- 0.18 mU/kg x min insulin infusion reduced N and 3-MH loss after a time lag of 12 h. The peak decrease in body N (-36%) and 3-MH loss (-38%) was reached during the first 12 h of glucose withdrawal period. Thereafter, during the following 12 h, the effect completely vanished confirming that it is therapy-dependent and that the metabolic environment of the patients did not change during the three days study period. CONCLUSION: 24 h glucose withdrawal reduces N and 3-MH loss injured patients, the drug-like effect is maintained during the first 12 h of withdrawal and thereafter disappears. The study suggests that at least a 24 h study period is necessary when planning studies exploring energy-protein metabolism relationship in injured patients, and, again 24 h before changing protocol in a crossover study.

Effect of infusion and withdrawl of glucose and insulin on gas exchange in injured ventilated patients.

PURPOSE: To evaluate the effect induced on gas exchange and on urea excretion by glucose and insulin infusion in injured patients. The magnitude and time necessary for the full development of the metabolic effect were investigated. METHODS: Six injured patients were investigated. During the first 24 hours, the fasting period, patients received 1 mg/kg*min of glucose; during the second 24 hours, the treatment period, infusion was increased to about the 95% of the energy production rate; during the last 8 hours, (stop period) the infusion rate was again set to 1 mg/kg*min. Gas exchange was determined in two consecutive 12-hour series, for 30 minutes every hour, either during a stabilized treatment or after its variation. Urea excretion was determinated on 4-hour samples. RESULTS: With respect to the fasting period, during the last 4 hours of the treatment period, the energy production rate did not vary; urea excretion (-25%) and oxygen consumption (-9%) decreased significantly. Carbon dioxide production (+16%), total respiratory quotient, and minute ventilation (+5%) increased significantly. Carbon dioxide production varied linearly with time (glucose infusion +1.74 mL/min*m2*h, P < .05; glucose withdrawal -1.89 mL/min*m2*h, P < .01). Minute ventilation decreased only during the withdrawal period by 65 mL/min*m2*h (P < .05). CONCLUSIONS: The infusion of glucose and insulin, in an amount slightly lower than the metabolic expenditure, leads to a consistently reduced amino acid catabolism and to a decreased oxygen consumption, without affecting energy requirements. Although it leads to an increase of carbon dioxide production, the measured change is so small and slow that it is not harmful unless there is severe respiratory insufficiency.

[Determination of the best amino acid input after orthotopic liver transplantation]. / Determinazione dell'apporto aminoacidico ottimale dopo trapianto ortotopico di fegato.

Ten-three patients were investigated during the early postoperative phase after orthotopic liver transplantation to assess the adequacy of the amino acid (AA) supply during both parenteral (days 1-5) and enteral (days 6-9) nutrition. Plasma AA profile was determined preoperatively, on day 4 and 5 during TPN and on day 8 and 9 during EN, urea production rate was measured every day. Calories input was 28 kcal.kg-.day as glucose, nitrogen intake was 0.25 g.kg- day, supplying individual AA on the basis of previous studies. Urea nitrogen production during TPN (9-11 gN/m2.day) outlines the ability of the transplanted liver to manage the overall nitrogen load. Individual AA plasma profile was considered the expression of an adequate input when comprised between 1 and 1.5 times the normal value, in this respect we obtained adequate levels of all essential AAs. Particularly phenylalanine, methionine and branched chain AA, critical during liver failure, were kept in this range by supplying 68, 48 and 500 mg.kg-1.day. According to AA profile the supply of cystine and tyrosine (conditionally essential AAs), and of histidine, taurine, proline and serine could be safely increased. Not given dispensable AAs (glutamine, asparagine, citrulline and alfa amino butyric) showed a plasma level below the norm and should be added to the diet.

[Changes in the plasma amino acid profile in critically ill patients during total parenteral therapy]. / Variazioni del profilo aminoacidico plasmatico in pazienti critici durante terapia parenterale totale.

The aim of this study was to evaluate the kinetics of arterial plasma amino acid profile during the first 48 h of clinical TPN in order to assess the time necessary to reach the steady-state condition during infusion. Each patient was treated with one of three different amino acid solutions yielding, in the same nitrogen intake, different intakes of individual amino acids. We found four different kinetics for the administered amino acids: an increase of plasma levels immediately after the start of the TPN with no variations during the steady period; the same trend with the steady-state obtained after 6-24 h of TPN infusion; no influence at all; a decrease of fasting plasma levels with the steady-state attained variably during the study period. Each given amino acid showed a different trend partly depending on the supply, suggesting that the steady-state was reached sooner for most amino acids, when the supply was larger. With lower intakes, plasma levels were unaffected or decreased. We conclude that in critically ill patients at least 24 h are needed to obtain stable arterial plasma amino acid concentration during TPN with adequate intakes of amino acid. Knowledge offers the possibility for a quick and accurate assessment of the adequacy of a given preparation (tailored for critically ill patients), it reduces the time span of the study and, as a consequence, the influence of varied metabolic conditions.

Basal energy production rate and substrate use in stable cirrhotic patients.

The basal energy production rate was measured using indirect calorimetry in 25 stable cirrhotic patients and 10 controls of comparable age. The endogenous substrate oxidation was also calculated by measuring urinary nitrogen excretion. The energy production rate was similar in cirrhotic patients and controls. The origins of liver disease and the degree of liver damage did not seem to influence the energy production rate. On the other hand, in cirrhotic patients, as in controls, a significant correlation was present between the energy production rate and parameters of body size, such as body weight and fat-free mass. As a consequence, cirrhotic patients with poor nutritional status, with a reduced fat-free mass, showed a lower energy production rate. The measured energy production rate was compared with the resting energy expenditure estimated by formulas commonly used in healthy individuals. The good agreement found between the measured energy production rate and calculated energy expenditure suggests that these formulas may be applied to stable cirrhotic patients in clinical practice. In cirrhotic patients, the oxidation of endogenous fat is the main contributor to basal energy production rate. The fat oxidation rate does not appear to be influenced by the hormonal pattern found in the cirrhotic patients. However, a significant correlation was present between fat oxidation and plasma free fatty acid levels. This confirms that the prevalent fat use in cirrhotic patients is supported by the greater availability of fat-derived substrates.