Sample-size determination and adherence in randomised controlled trials published in anaesthetic journals

Background: Sample-size calculations are critical to ensure that randomised control trials return robust and reliable results. The estimated treatment effects used in these calculations is often significantly different from the actual treatment effect and can dramatically impact trial validity. Methods: This study examined sample-size calculations in randomised controlled trials designed to show superiority between two-arm parallel groups with a single primary outcome that were published in the top five anaesthetic journals for 2014 (as per Thomson Reuters impact factors). In particular, it sought to determine treatment effect estimations used in a priori sample-size calculations and compare them with actual treatment effects. Results: A PubMed search identified 209 possible articles; 52 were drawn for full text review; and 28 were included in the final analysis. The relative difference between expected and actual event rates was greater than 20% in 80% of trials and greater than 50% in 44% of trials. Conclusions: Unrealistic assumptions of treatment effects in randomised controlled trials published in anaesthesia journals are common. Trial sample sizes should be calculated thoughtfully and realistically and should be fully reported in both trial protocols and publications. Researchers should be aware of the opportunity cost as well as the possible dangers to patients when unrealistic assumptions are made. Where possible researchers should collaborate to achieve meaningful trial sample sizes to ensure robust clinical findings

Concept and early development of solid-phase peptide synthesis.

There are several reasons for the success of the solid-phase approach to peptide synthesis. The first is the ease of the procedure, the acceleration of the overall process, and the ability to achieve good yields of purified products. The second was the unanticipated discovery of many new biologically active peptides and the expanded need for synthetic peptides to help solve problems in virtually all disciplines of biology. In many cases, the solid-phase technique has been the method of choice. This approach, of course, does not replace the classic solution synthesis methods, but rather supplements them. The choice of techniques depends on the objectives of the synthesis. When carefully worked out, the solution methods can give high yields of highly purified products in large quantities. Many superb syntheses of active peptides have been achieved in this way. The solid-phase method has also yielded many large active peptides. It is particularly useful when large numbers of analogs, in relatively small quantities, are required as in structure-function studies on hormones, growth factors, antibiotics, and other biologically active peptides or for determining the antigenic epitopes of proteins. In addition, it has on occasion been scaled up for production of kilogram quantities. One of the unique uses of solid-phase synthesis has been the synthesis of peptide libraries. Most of the work on this new field in which thousands or millions of peptides are prepared simultaneously has been by solid-phase methods. This new technique is proving to be of great practical importance in rapid drug discovery of peptide, peptide mimetic, and nonpeptide compounds. Developments in screening methods now allow the examination of large numbers of compounds, and active products with structures unpredictable from natural product sequences are being found in this way. The properties of the solid-phase system, the changes in the chemistry, and the applications of the technique to biological problems are discussed in detail in subsequent articles of this volume.

Increasing mean skin temperature linearly reduces the core-temperature thresholds for vasoconstriction and shivering in humans.

BACKGROUND: The contribution of mean skin temperature to the thresholds for sweating and active precapillary vasodilation has been evaluated in numerous human studies. In contrast, the contribution of skin temperature to the control of cold responses such as arteriovenous shunt vasoconstriction and shivering is less well established. Accordingly, the authors tested the hypothesis that mean skin and core temperatures are linearly related at the vasoconstriction and shivering thresholds in men. Because the relation between skin and core temperatures might vary by gender, the cutaneous contribution to thermoregulatory control also was determined in women. METHODS: In the first portion of the study, six men participated on 5 randomly ordered days, during which mean skin temperatures were maintained near 31, 34, 35, 36, and 37 degrees C. Core hypothermia was induced by central venous infusion of cold lactated Ringer's solution sufficient to induce peripheral vasoconstriction and shivering. The core-temperature thresholds were then plotted against skin temperature and a linear regression fit to the values. The relative skin and core contributions to the control of each response were calculated from the slopes of the regression equations. In the second portion of the study, six women participated on three randomly ordered days, during which mean skin temperatures were maintained near 31, 35, and 37 degrees C. At each designated skin temperature, core hypothermia sufficient to induce peripheral vasoconstriction and/or shivering was again induced by central venous infusion of cold lactated Ringer's solution. The cutaneous contributions to control of each response were then calculated from the skin- and core-temperature pairs at the vasoconstriction and shivering thresholds. RESULTS: There was a linear relation between mean skin and core temperatures at the response thresholds in the men: r = 0.90 +/- 0.06 for vasoconstriction and r = 0.94 +/- 0.07 for shivering. Skin temperature contributed 20 +/- 6% to vasoconstriction and 19 +/- 8% to shivering. Skin temperature in the women contributed to 18 +/- 4% to vasoconstriction and 18 +/- 7% to shivering, values not differing significantly from those in men. There was no apparent correlation between the cutaneous contributions to vasoconstriction and shivering in individual volunteers. CONCLUSIONS: These data indicate that skin and core temperatures contribute linearly to the control of vasoconstriction and shivering in men and that the cutaneous contributions average approximately 20% in both men and women. The same coefficients thus can be used to compensate for experimental skin temperature manipulations in men and women. However, the cutaneous contributions to each response vary among volunteers; furthermore, the contributions to the two responses vary within volunteers.

Propofol linearly reduces the vasoconstriction and shivering thresholds.

BACKGROUND: Skin temperature is best kept constant when determining response thresholds because both skin and core temperatures contribute to thermoregulatory control. In practice, however, it is difficult to evaluate both warm and cold thresholds while maintaining constant cutaneous temperature. A recent study shows that vasoconstriction and shivering thresholds are a linear function of skin and core temperatures, with skin contributing 20 +/- 6% and 19 +/- 8%, respectively. (Skin temperature has long been known to contribute approximately 10% to the control of sweating). Using these relations, we were able to experimentally manipulate both skin and core temperatures, subsequently compensate for the changes in skin temperature, and finally report the results in terms of calculated core-temperature thresholds at a single-designated skin temperature. METHODS: Five volunteers were each studied on 4 days: (1) control; (2) a target blood propofol concentration of 2 micrograms/ml; (3) a target concentration of 4 micrograms/ml; and (4) a target concentration of 8 micrograms/ml. On each day, we increased skin and core temperatures sufficiently to provoke sweating. Skin and core temperatures were subsequently reduced to elicit peripheral vasoconstriction and shivering. We mathematically compensated for changes in skin temperature by using the established linear cutaneous contributions to the control of sweating (10%) and to vasoconstriction and shivering (20%). From these calculated core-temperature thresholds (at a designated skin temperature of 35.7 degrees C), the propofol concentration-response curves for the sweating, vasoconstriction, and shivering thresholds were analyzed using linear regression. We validated this new method by comparing the concentration-dependent effects of propofol with those obtained previously with an established model. RESULTS: The concentration-response slopes for sweating and vasoconstriction were virtually identical to those reported previously. Propofol significantly decreased the core temperature triggering vasoconstriction (slope = -0.6 +/- 0.1 degrees C.micrograms-1.ml-1; r2 = 0.98 +/- 0.02) and shivering (slope = -0.7 +/- 0.1 degrees C.micrograms -1.ml-1; r2 = 0.95 +/- 0.05). In contrast, increasing the blood propofol concentration increased the sweating threshold only slightly (slope = 0.1 +/- 0.1 degrees C.micrograms -1.ml-1; r2 = 0.46 +/- 0.39). CONCLUSIONS: Advantages of this new model include its being nearly noninvasive and requiring relatively little core-temperature manipulation. Propofol only slightly alters the sweating threshold, but markedly reduces the vasoconstriction and shivering thresholds. Reductions in the shivering and vasoconstriction thresholds are similar; that is, the vasoconstriction-to-shivering range increases only slightly during anesthesia.

Optimal duration and temperature of prewarming.

BACKGROUND: Core hypothermia developing immediately after induction of anesthesia results largely from an internal core-to-peripheral redistribution of body heat. Although difficult to treat, redistribution can be prevented by prewarming. The benefits of prewarming may be limited by sweating, thermal discomfort, and efficacy of the warming device. Accordingly, the optimal heater temperature and minimum warming duration likely to substantially reduce redistribution hypothermia were evaluated. METHODS: Sweating, thermal comfort, and extremity heat content were evaluated in seven volunteers. They participated on two study days, each consisting of a 2-h control period followed by 2 h of forced-air warming with the heater set on "medium" (approximately 40 degrees C) or "high" (approximately 43 degrees C). Arm and leg tissue heat contents were determined from 19 intramuscular needle thermocouples, ten skin temperatures, and "deep" foot temperature. RESULTS: Half the volunteers started sweating during the second hour of warming. None of the volunteers felt uncomfortably warm during the first hour of heating, but many subsequently did. With the heater set on "high," arm and leg heat content increased 69 kcal during the first 30 min of warming and 136 kcal during the first hour of warming, representing 38% and 75%, respectively, of the values observed after 2 h of warming. The increase was only slightly less when the heater was set to "medium." CONCLUSIONS: Neither sweating nor thermal discomfort limited heat transfer during the first hour of warming. Thirty minutes of forced-air warming increased peripheral tissue heat content by more than the amount normally redistributed during the first hour of anesthesia. The large increase in arm and leg heat content during prewarming thus explains the observed efficacy of prewarming.

Structure-immunogenicity relationship of melittin, its transposed analogues, and D-melittin.

Melittin, a 26-residue bee venom peptide, is known to induce murine Abs specific for its hydrophilic C-terminus of residues 20-26 and T cell responses specific for its hydrophobic mid-region of residue 11-19. Synthetic melittin analogues with transposed sequences of Ac(21-26) (1-20) and Ac(26-21) (1-20) are found to induce murine Abs specific for the transposed peptide segment and to induce T cell responses that are cross-reactive with melittin. Compared with melittin, the transposed melittin analogues are weaker immunogens and have lower hemolytic activities, lower helical contents, and a lower degree of association in micelles. A melittin analogue with a lactoside group at its C-terminus was found to induce lactoside-specific murine Abs. Present studies show that another analogue with a lactoside group at its N-terminus induces only Abs specific for the C-terminal region of melittin, and no lactoside-specific Abs are detected. These immunochemical observations suggest that the immunogenicity of melittin or its analogues is a consequence of its binding to cell membranes with subsequent oligomer formation in lipid bilayers. Apparently melittin or its analogues bind to cell membrane in an asymmetric manner with the exposed and the buried segments functioning as B and T cell epitopes, respectively. D-melittin is non-immunogenic in mice, although D-melittin has the same hemolytic activity as melittin. This finding may be correlated with the known resistance of D-melittin to proteolysis and hence to processing for Ag presentation to T lymphocytes.

The pupillary light reflex. Effects of anesthetics and hyperthermia.

BACKGROUND: The pupillary light reflex often is evaluated in the perianesthetic period to assess drug effects and brainstem function. Mild hypothermia alone or combined with isoflurane does not impair pupillary responses. Although perioperative hyperthermia is less common than hypothermia, abnormal increases in core temperature remain an important thermal disturbance. Accordingly, the pupillary effects of hyperthermia alone and hyperthermia combined with isoflurane and enflurane were evaluated. Additionally, the effects of nitrous oxide on pupillary responses were determined. METHODS: The pupillary light reflex was evaluated in 31 non-surgical volunteers participating in concurrent thermoregulatory studies. Pupillary reflexes were quantified using a portable infrared pupillometer during (1) hyperthermia alone (n = 9), (2) hyperthermia with 0.8% and 1.2% end-tidal isoflurane (n = 8), (3) hyperthermia with 1.7% end-tidal enflurane (n = 5), and (4) inhalation of 60% N2O (n = 9). RESULTS: Mild hyperthermia alone (core temperature 38.5 +/- 0.3 degrees C) produced no clinically significant change in the pupillary light reflex. Pupillary responses were decreased markedly with 0.8% isoflurane, 1.2% isoflurane, and 1.7% enflurane when the volunteers were normothermic. Mild hyperthermia combined with isoflurane or enflurane dilated the pupil and increased the amplitude of the light reflex. Sixty-percent nitrous oxide decreased the pupillary reflex only 26 +/- 4%. CONCLUSIONS: Anesthetic-induced inhibition of the pupillary response to light is reversed partially by core hyperthermia. In contrast to enflurane and isoflurane, 60% N2O has little effect on the pupil.

Pupillary response to noxious stimulation during isoflurane and propofol anesthesia.

We studied the effects of noxious stimuli on arterial blood pressure, heart rate, pupil size, and the pupillary light reflex in 13 volunteers anesthetized with either isoflurane or propofol. Those given isoflurane (n = 8) were anesthetized twice, in a randomly selected order, once at an end-tidal concentration of 0.8% and once at 1.2%. An intense noxious stimulus was provided by electrical stimulation applied to skin of the abdominal wall (65-70 mA, 100 Hz). Hemodynamic values and pupillary responses were recorded immediately before stimulation and at 15-60-s intervals during 8 subsequent min. In the volunteers given isoflurane (both concentrations), stimulation significantly increased pupil size (265 +/- 44%) and the amplitude of the light reflex (233 +/- 23%). In contrast, mean heart rate and systolic blood pressure increased only 19 +/- 7% and 13 +/- 7% after stimulation. Five additional volunteers were anesthetized twice with propofol (approximately 3 micrograms/mL plasma concentration) and 60% nitrous oxide. The same electrical stimulus was applied, and hemodynamic and pupillary measurements were obtained. During one propofol anesthetic, an esmolol infusion (100 micrograms.kg-1 x min-1) was started 10 min before stimulation to determine whether this agent would blunt the pupillary response. The pupillary light reflex increased more than 200% during both propofol anesthetics with or without esmolol; once again, heart rate and blood pressure changed little. We conclude that with these experimental conditions, the pupil is a more sensitive measure of noxious stimulation than the commonly used variables of arterial blood pressure and heart rate.

Leg heat content continues to decrease during the core temperature plateau in humans anesthetized with isoflurane.

BACKGROUND: Sufficient hypothermia during anesthesia provokes thermoregulatory responses, but the clinical significance of these responses remains unknown. Nonshivering thermogenesis does not increase metabolic heat production in anesthetized adults. Vasoconstriction reduces cutaneous heat loss, but the initial decrease appears insufficient to cause a thermal steady state (heat production equaling heat loss). Accordingly, the authors tested the hypotheses that: 1) thermoregulatory vasoconstriction prevents further core hypothermia; and 2) the resulting stable core temperature is not a thermal steady state, but, instead, is accompanied for several hours by a continued reduction in body heat content. METHODS: Six healthy volunteers were anesthetized with isoflurane (0.8%) and paralyzed with vecuronium. Core hypothermia was induced by fan cooling, and continued for 3 h after vasoconstriction in the legs was detected. Leg heat content was calculated from six needle thermocouples and skin temperature, by integrating the resulting parabolic regression over volume. RESULTS: Core temperature decreased 1.0 +/- 0.2 degrees C in the 1 h before vasoconstriction, but only 0.4 +/- 0.3 degrees C in the subsequent 3 h. This temperature decrease, evenly distributed throughout the body, would reduce leg heat content 10 kcal. However, measured leg heat content decreased 49 +/- 18 kcal in the 3 h after vasoconstriction. CONCLUSIONS: These data thus indicate that thermoregulatory vasoconstriction produces a clinically important reduction in the rate of core cooling. This core temperature plateau resulted, at least in part, from sequestration of metabolic heat to the core which allowed core temperature to remain nearly constant, despite a continually decreasing body heat content.

Thermoregulatory responses to hyperthermia during isoflurane anesthesia in humans.

The authors tested the hypotheses that isoflurane anesthesia increases the threshold for sweating but minimally decreases the gain (sensitivity) or maximum intensity of this response and that thermoregulatory responses to hyperthermia are similar in anesthetized men and women. Sweating in response to core hyperthermia was studied in five men and five women during 0, 0.8, and 1.2% end-tidal isoflurane anesthesia. Thigh sweating was quantified by measuring gas flow, relative humidity, and temperature passing over a known surface area. The distal esophageal temperature triggering sweating was considered the sweating threshold, and gain was defined as the core temperature increment required to increase sweating rate from 25 to 75% of maximum observed intensity. The sweating threshold increased linearly with isoflurane concentration from 36.6 +/- 0.1 to 38.1 +/- 0.1 degrees C in the men and from 37.1 +/- 0.3 to 38.3 +/- 0.2 degrees C in the women. The thresholds were significantly higher in women than in men. Gain and maximum sweating intensities were similar at each anesthetic concentration and in men and women. These data indicate that isoflurane anesthesia significantly increases the threshold triggering thermoregulatory sweating but that gain and maximum sweating rate are relatively well preserved.

Thermoregulatory vasoconstriction during propofol/nitrous oxide anesthesia in humans: threshold and oxyhemoglobin saturation.

To determine the thermoregulatory effects of propofol and nitrous oxide, we measured the threshold for peripheral vasoconstriction in seven volunteers over a total of 13 study days. We also evaluated the effect of vasoconstriction on oxyhemoglobin saturation (SpO2). Anesthesia was induced with an intravenous bolus dose of propofol (2 mg/kg), followed by an infusion of 180 micrograms.kg-1 x min-1 for 15 min, and maintained with 60% nitrous oxide and propofol (80-160 micrograms.kg-1 x min-1). Central and skin surface temperatures and SpO2 (using two different pulse oximeters) were measured continuously; plasma propofol concentrations and arterial PO2 were measured at 15-min intervals. Volunteers were cooled with a circulating water blanket until definitive peripheral vasoconstriction was detected. The tympanic membrane temperature triggering vasoconstriction was considered the thermoregulatory threshold. Vasoconstriction developed on seven study days during propofol/nitrous oxide anesthesia at a central temperature of 33.3 +/- 1.0 degrees C (mean +/- SD) and plasma propofol concentration of 3.9 +/- 1.1 micrograms/mL. The thresholds during anesthesia were significantly lower than those during the control period (36.7 +/- 0.3 degrees C), but the correlation between plasma propofol concentrations and vasoconstriction thresholds was poor. On the remaining six study days, vasoconstriction did not develop despite central temperatures ranging from 32.1 to 32.7 degrees C. Corresponding propofol concentrations were 4.1-10.9 micrograms/mL. These data suggest that anesthesia with propofol, in typical clinical concentrations, and 60% nitrous oxide substantially inhibits thermoregulatory vasoconstriction. Vasoconstriction increased SpO2 by approximately 2% without a significant concomitant change in PO2. The observed increase in SpO2 probably reflects decreased transmission of arterial pulsations to venous blood in the finger.

Ocular hypothermia depresses the human pupillary light reflex.

Twelve human volunteers were studied to determine the effect of eye surface cooling on the parameters of the light reflex. Surface cooling resulted in a 38% decrease in the maximum constriction velocity and a 36% decrease in the maximum redilation velocity. These changes were thought to be the result of a "sluggish" response of the pupillary sphincter.

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease.

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.

The antifungal activity of carrier peptides, L-arginyl-X-L-phenylalanine, containing amino acid antagonists or atypical non-biogenic D-amino acids in the central position.

Eleven analogues of L-arginyl-D-allo-threonyl-L-phenylalanine, a naturally occurring peptide with antifungal activity, were synthesized. Two tripeptides of the form L-arginyl-X-L-phenylalanine (X = p-F-DL-phenylalanine or m-F-DL-tyrosine) inhibited Aspergillus fumigatus and Aspergillus flavus. In comparison with the free antagonists, the tripeptide-bound antagonists were more active against Candida-albicans-isolates which means that the amino acid sequence may serve as carrier function: it enhances delivery or uptake of the antimetabolite.