Effect of cell density on hormonal secretion from human pituitary adenomas in vitro.

UNLABELLED: Cell density effects were investigated on tumorous hormonal secretion from 10 pituitary adenomas: 3 somatotrophinomas secreting GH and PRL; 7 gonadotrophinomas, 3 co-secreted both FSH and LH, all 7 secreted LH. Enzymatically dispersed tissue was plated out in 24-well plates at 5 x 10(5), 10(5), 5 x 10(4) and 10(4) cells/well in serum-free media. Media were collected weekly for 2 weeks. RESULTS: In 3 of 3 somatotrophinomas, GH and PRL secretion was higher (p < 0.05) at both week 1 and 2 from 10(4) cells/well, but similar at other cell densities. In all 3 gonadotrophinomas, the FSH secretory rate was highest at 5 x 10(5) cells/well which fell as cell density decreased. Conversely, in 7 of 7 gonadotrophinomas the LH secretory rate was highest at 10(4) cells/well (p < 0.01) which fell as cell density increased. CONCLUSION: These data suggest that paracrine factors may modulate tumorous GH, PRL, FSH and LH secretion, and show that FSH and LH secretion vary inversely as cell density increases.

Gonadal status is an important determinant of bone density in acromegaly.

OBJECTIVE: To measure bone mineral density (BMD) in patients with acromegaly and to look for clinical features which may explain previously reported discrepant results. DESIGN: Prospective case controlled observational study. PATIENTS: 18 patients with acromegaly (seven women, 11 men; mean age 53.2 +/- 3.5 years). Eight patients had active disease and 10 had controlled acromegaly. Growth hormone and insulin-like growth factor-1 (IGF-1) concentrations were measured to assess disease activity. MEASUREMENTS: BMD was measured at four sites on the lumbar spine, three at the femoral neck and at Ward's triangle using a Hologic dexa scanner. Results were compared to a locally determined control population (n = 1800). In eight patients with active acromegaly, urinary free pyridinoline and deoxypyridinoline, and serum osteocalcin, propeptide of type 1 procollagen, vitamin D, 1000 h parathyroid hormone, bone specific alkaline phosphatase, calcium and phosphate concentrations were measured before and after 6 months treatment with octreotide. RESULTS: The mean BMD for all acromegalic patients was not significantly different from the control population except at the femoral neck where it was increased (P = 0.05). At all sites, the BMD of patients who had been hypogonadal (n = 12/18) was significantly lower (P < 0.05-0.01) than that of patients who had been eugonadal (n = 6/18). BMD for hypogonadal patients was lower than the control population at Ward's triangle (P = 0.03). Eugonadal acromegalic patients had BMD greater than non-acromegalic controls at all sites. Patients with controlled acromegaly had a higher BMD than non-acromegalic controls, but there were no differences in BMD between patients with active and controlled acromegaly. Serum IGF-1 concentrations decreased from 64.5 +/- 5.1 nmol/l to 37.5 +/- 6.9 nmol/l (P = 0.02) after 6 months treatment with octreotide, but there was no change in any of the biochemical markers of bone turnover. CONCLUSIONS: Eugonadal acromegalic patients have increased lumbar spine and femoral neck BMD compared to hypogonodal acromegalic patients and the general population, but it is reduced if patients have been hypogonadal.

Conservative management of azoospermia following steroid abuse.

As well as athletes and competitive body builders, recreational body builders attending gymnasia are known to abuse anabolic steroids, using doses from 10- to 40-fold above physiological levels. Androgenic steroids induce hypogonadotrophic hypogonadism with associated azoospermia, leading to infertility. Little literature exists on the treatment of steroid-induced azoospermia following the cessation of abuse. We present four cases of steroid-induced azoospermia, its conservative management and eventual return of normal semen density.

Identification of proliferating human anterior pituitary adenoma cells in vitro.

A method to determine whether dispersed human anterior pituitary adenoma cells proliferate in mixed culture was developed. Fifteen pituitary adenomas were dispersed enzymatically to single cells, following which twelve were double immunostained after eight days. Proliferating cells were identified immunologically following one hour of bromo-deoxyuridine incorporation. Adenoma cells were subsequently identified with an anti-neuron-specific enolase antibody system. A time course of bromo-deoxyuridine labelling was performed on three nonfunctional adenomas over a four day period, with bromo-deoxyuridine being added to cultures at one hour, 24 hours and four days prior to immunostaining. Double immunolabelled cells were unambiguously identified by a dark brown nucleus surrounded by red cytoplasm. Eight out of 12 pituitary adenomas (two prolactinomas, three nonfunctional, three growth hormone secreting) showed an increased bromo-deoxyuridine labelling index (range 0.1%-1.4%). Bromo-deoxyuridine incorporation over four days showed an increase in bromo-deoxyuridine from 0.02%, 0.03% and 3.3% at one hour to 10.1%, 1.3% and 5.0% at four days, respectively, but evidence of mitosis was scant. This study shows that pituitary adenomas may proliferate in vitro and that this double immunostaining method may be used as an in vitro proliferation assay in a mixed cell population.

A comparison of proliferation indices in human anterior pituitary adenomas using formalin-fixed tissue and in vitro cell culture.

The authors compared detection methods for cell proliferation in human anterior pituitary adenomas using histological sections and dispersed cell culture. After tumor cells had been grown for 4 days in dispersed culture, bromodeoxyuridine (BUdR), proliferating cell nuclear antigen (PCNA), and Ki-67 were compared by double immunostaining and contrasted with single staining of PCNA and Ki-67 indices in the corresponding histological sections from 12 human pituitary adenomas. In vitro, the BUdR labeling index was positive in six of 12 tumors (range < 0.1-5.1%), 10 of 12 tumors were PCNA-positive (range < 0.1-100%), and Ki-67 was positive in 10 of 12 adenomas (range < 0.1-8%). In vitro, BUdR and Ki-67 gave similar proliferative indices for 10 of 12 adenomas. In vivo, the PCNA labeling index was positive in 12 of 12 adenomas (range 0.9-95%) and Ki-67 was positive in 11 of 12 adenomas (range < 0.1-2%). Tumors with a labeling index less than 0.1% were considered to be negative for proliferation. High PCNA values were found in vitro and in vivo, whereas Ki-67 labeling indices were similar in vitro and in vivo for nine of 12 adenomas. It is concluded that Ki-67 proliferative indices in vivo reflect those found in vitro, at least after 4 days in dispersed culture, but that PCNA overestimates pituitary adenoma proliferation in histological sections as well as in dispersed culture.

Apoptosis and p53 suppressor gene protein expression in human anterior pituitary adenomas.

Human anterior pituitary adenomas proliferate and express the p53 tumour suppressor gene protein, but it is not known if apoptosis (programmed cell death) occurs. Therefore, the detection of apoptosis was undertaken in tumorous human anterior pituitary tissue and compared with p53 protein expression, tumour type and tumour size. Apoptosis (detected by the in situ end labelling technique) and p53 suppressor gene protein (detected by DO.1-antibody immunocytochemistry) were determined in formalin-fixed and paraffin-embedded tissue from 37 human pituitary adenomas (2 macroprolactinomas, 9 somatotrophinomas and 26 non-functioning adenomas). Two normal anterior pituitaries were also included in this study. Pre-operative tumour size was scored 1 to 4 from magnetic resonance imaging radiology. Apoptosis was found in 7 of 29 tumours (24%), 11% of somatotrophinomas and 33% of non-functioning adenomas, although this difference was not significant. The p53 tumour suppressor protein was found in 7 of 31 tumours (23%), 33% of somatotrophinomas and 19% of non-functioning adenomas. Apoptosis and p53 protein expression were not found in normal anterior pituitary. In conclusion, apoptosis occurs in human anterior pituitary adenomas, but no significant association was found between apoptosis and p53 protein expression, tumour type or tumour size.

Cytokine expression in human anterior pituitary adenomas.

OBJECTIVE: There is increasing evidence for the role of cytokines in pituitary differentiated function and tumorigenesis, but the spectrum of cytokines found in the pituitary is unknown. Therefore profiles of cytokine expression were determined in different human anterior pituitary adenoma sub-types. DESIGN: The reverse transcriptase-linked polymerase chain reaction (PCR) was used to identify the presence of cytokine mRNA within human pituitary adenomas. PATIENTS: Seventeen pituitary adenoma biopsies removed at transsphenoidal surgery were examined: 4 somatotrophinomas, 7 non-functional adenomas, 4 prolactinomas, one case of Cushing's disease and one case of Nelson's syndrome. MEASUREMENTS: RNA was extracted from each adenoma biopsy and reverse transcribed into cDNA. This was specifically amplified in a PCR using oligonucleotide primers complementary to each cytokine. The cytokines investigated were interleukin (IL)-I alpha, IL-I beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-alpha, TNF-beta and transforming growth factor (TGF)-beta 1, beta 2 and beta 3. The products of each PCR were visualized using agarose gel electrophoresis. RESULTS: All 17 adenomas expressed IL-8 transcripts, but no expression of IL-2, IL-5 or IL-7 was found. IL-6 was expressed in all 4 somatotrophinomas, 3 of 7 non-functional tumours, 2 of 4 prolactinomas and in the single case of Nelson's syndrome. At least one of the 3 isoforms of TGF-beta was found in all but 2 tumours; one prolactinoma and one non-functional adenoma. IL-1 alpha, IL-beta, IL-4, TNF-alpha and TNF-beta were expressed sporadically by individual adenomas. CONCLUSION: These data suggest that whilst IL-8 may be important, the local expression of the cytokines IL-2, IL-5 and IL-7 is not important in human anterior pituitary tumorigenesis.

The role of a single free beta-human chorionic gonadotrophin measurement in the diagnosis of early pregnancy failure and the prognosis of fetal viability.

The prospective controlled study investigated the concentrations of free beta-human chorionic gonadotrophin (HCG) subunit in 554 women with a singleton intrauterine or tubal pregnancy. They presented with vaginal bleeding and/or abdominal pain in the first 18 weeks of pregnancy. The control group comprised 156 women with musculoskeletal pain and no vaginal bleeding. Their pregnancies continued to term. The study group comprised 398 women (141 threatened-continuing pregnancies, 37 threatened-miscarriages, 185 non-continuing pregnancies and 35 tubal pregnancies). Free beta-HCG concentrations were significantly lower in the non-continuing, threatened-miscarriage and tubal pregnancy groups [mean 4.62, 6.50 and 4.27 ng/ml respectively; 95% confidence interval (CI) 3.75-5.69, 4.46-9.48 and 2.92-6.2 respectively] than in the control and threatened-continuing groups (mean 41.61 and 48.22 ng/ml respectively; 95% CI 34.53-50.13 and 42.03-55.32 respectively) (P < 0.001 in all cases). A cut-off value at 20 ng/ml was found to differentiate between the 'viable' (control and threatened-continuing) and the 'abnormal' (non-continuing, threatened-miscarriage and tubal) pregnancies, with 88.3% sensitivity and 82.6% positive predictive value. An excellent diagnostic and prognostic usability of free beta HCG was confirmed by a receiver operating characteristic curve plot. In conclusion, a single serum free beta-HCG measurement taken in early pregnancy is valuable in the immediate diagnosis of early pregnancy failure and the long-term prognosis of viability.

Septic patients in multiple organ failure can oxidize infused glucose, but non-oxidative disposal (storage) is impaired.

1. Patients suffering trauma and sepsis are insulin resistant, but no studies have specifically been made of patients suffering multiple organ failure. 2. We have studied exogenous glucose utilization in multiple organ failure using a combination of the hyperglycaemic glucose clamp and indirect calorimetry to quantify glucose utilization in multiple organ failure, partitioning it into oxidative and nonoxidative disposal (storage). 3. Fourteen septic patients with multiple organ failure were studied. APACHE II (Acute Physiological and Chronic Health Evaluation Mark II) scores on the day of the study ranged from 11 to 31 (median 16). Twenty percent D-glucose was infused and blood glucose was clamped at 12 mmol/l for 3 h. The results were compared with those obtained on seven healthy control subjects. 4. Glucose utilization and energy expenditure were similar in the two groups for the first 90 min of the clamp, after which glucose utilization and energy expenditure increased steadily in the control subjects but did not change in the patients. Respiratory exchange ratio rose in both groups; considered over the whole of the clamp period, respiratory exchange ratio was slightly lower in the patients than in the control subjects (P < 0.05) but not at any specific time point. Glucose oxidation rose in both groups but non-oxidative glucose disposal (storage) rose only in the control subjects. Glucose oxidation was slightly lower in the patients (P < 0.05) but not at any specific time point and there was no difference between the groups in the amount by which glucose oxidation increased. Non-oxidative disposal in the patients fell significantly (P < 0.01) over the course of the clamp and was significantly lower than in the control subjects (P < 0.01). 5. Growth hormone increased in response to glucose infusion in the patients but not in the control subjects. 6. Like patients suffering uncomplicated sepsis or trauma, patients with multiple organ failure are also insulin resistant. The defect appears to lie in an impairment of the ability to store glucose rather than oxidize it, and this may be due in part to the increase in growth hormone in patients with multiple organ failure.

The role of a single progesterone measurement in the diagnosis of early pregnancy failure and the prognosis of fetal viability.

OBJECTIVE: To assess the role of a single maternal serum progesterone measurement in the immediate diagnosis of early pregnancy failure and in the long term prognosis of fetal viability. DESIGN: A prospective comparative study of women presenting with vaginal bleeding and abdominal pain in early pregnancy. The comparison group was defined retrospectively as women who presented with abdominal pain without history of, or the subsequent occurrence of, vaginal bleeding and whose pregnancies continued to viability. The study groups were defined retrospectively as threatened-continuing, non-continuing (including blighted ovum, missed abortion, incomplete and complete abortion) and tubal pregnancy groups, according to the outcome of the pregnancies. SETTING: The emergency room at the gynaecology department of a teaching hospital. SUBJECTS: Four hundred and eighty-nine women presenting with singleton pregnancy, vaginal bleeding and/or abdominal pain in the first 18 weeks of pregnancy. The comparison group comprised 131 women without vaginal bleeding whose pregnancies continued to viability. The study group comprised 358 women with 148 threatened-continuing pregnancies, 175 non-continuing and 35 tubal pregnancies. INTERVENTIONS: A 10 ml blood sample was taken and pelvic ultrasonography was performed at presentation. Otherwise, conventional management was used. MAIN OUTCOME MEASURES: Progesterone levels were interpreted in accordance with the outcome of the pregnancy: comparison, threatened-continuing, non-continuing or tubal. Viability was defined as 28 weeks or more weeks of gestation. RESULTS: Progesterone levels were significantly lower in the non-continuing and tubal pregnancy groups than in the comparison and threatened-continuing groups (P < 0.001 in all cases). A cut-off level at 45 nmol/1 was found to differentiate between the viable (comparison and threatened-continuing) pregnancies and the abnormal (non-continuing and tubal) pregnancies with 87.6% sensitivity and 87.5% specificity. CONCLUSIONS: A single serum progesterone measurement taken in early pregnancy is valuable in the immediate diagnosis of early pregnancy failure and the long term prognosis of viability.

Manipulation of substrate utilization with somatostatin in patients with secondary multiple organ dysfunction syndrome.

OBJECTIVES: To determine whether glucose utilization and metabolic substrate (glucose and fat) oxidation could be manipulated in patients with secondary multiple organ dysfunction syndrome. DESIGN: Prospective study. SETTING: Intensive care units (ICU) of two university hospitals. PATIENTS: Eight adults free of hepatic disease and hemodynamically stable at the time of study, but with failed respiratory and gastrointestinal systems, who thus required mechanical ventilation and intravenous nutrition. INTERVENTIONS: Patients were infused with 20% dextrose through central venous cannulas at rates that increased and maintained (clamped) their plasma glucose concentration at 216 mg/dL (12 mmol/L) for 3 hrs. Somatostatin was infused continuously during the second and third hours of the clamp to reduce plasma concentrations of endogenous insulin and glucagon. Exogenous insulin was administered together with somatostatin during the third hour to restore basal insulin concentrations. Energy expenditure was measured by indirect calorimetry throughout the study and blood samples were withdrawn regularly for determination of metabolite and hormone concentrations. Main statistical comparisons were made between the baseline data (first hour of the study) and data collected during the second and third hours of the clamp. MEASUREMENTS AND MAIN RESULTS: Plasma glucagon concentrations were reduced by nearly 50% (p < .05) toward the end of the study, whereas no significant changes in plasma concentrations of cortisol or growth hormone occurred. Energy expenditure did not change significantly at any time during the clamp procedure. Glucose utilization (6.1 mg/kg/min [34 mumol/kg/min]) during the first hour of the hyperglycemic clamp, decreased by 53% (p < .05) with the infusion of somatostatin during the second hour of the clamp. However, once exogenous insulin was infused during the third hour, glucose utilization increased by 55% (p < .05) when compared with the baseline (hour 1) rate. Glucose oxidation was nearly doubled during the third hour of the study when compared with oxidation rates during the first and second hours. Fat oxidation decreased steadily during the 3-hr clamp. CONCLUSIONS: Glucagon has a significant inhibitory effect on glucose utilization during intravenous glucose infusion in the multiple organ dysfunction syndrome patient. Pharmacologic intervention with somatostatin and insulin (physiologic dose) can facilitate glucose utilization and oxidation in these patients. Further investigations are needed to determine whether long-term alteration of glucose and fat metabolism would be beneficial in the patient with secondary multiple organ dysfunction syndrome.

Effects of basic fibroblastic growth factor on the function and proliferation of human clinically non-functional pituitary adenomas which secreted glycoprotein hormones in vitro.

The effects of human recombinant basic fibroblastic growth factor (bFGF) on the secretion, viability, proliferation, attachment and morphology of ten dispersed human clinically non-functional (NF) adenomas were examined in vitro. Four clinically NF adenomas secreting FSH and/or LH in vitro were unaffected by 10 nM bFGF over a 4-h period. Over 4 days 10 nM bFGF stimulated LH secretion (66% and 72%, P < 0.01) from two out of seven clinically NF adenomas secreting LH, whilst FSH (three tumours) and alpha-subunit secretion (three tumours) were unaffected. One adenoma co-secreting LH and alpha-subunit and one secreting LH alone were studied over 21 days; LH secretion fell progressively, but the decline was significantly less (P < 0.05) with bFGF (10 nM) treatment after 14 and 21 days in both adenomas, whilst the fall in alpha-subunit secretion was unaffected by bFGF treatment. A 24-h GnRH test performed at the start and end of the 21-day period in one of these tumours showed an increase in both basal and stimulated LH secretion in the bFGF-treated group over control (124%, P < 0.001). There was no effect of bFGF (10 nM) on viability, S-phase proliferation, attachment or morphology of adenoma cells over a 4-day period. These results suggest that bFGF has a role in tumorous LH secretion from these adenomas, but is not mitogenic (at least over 4 days) and is without effect on other parameters of in vitro differentiated function.

The long-term stability in whole blood of 14 commonly-requested hormone analytes.

Concentrations of 14 commonly-requested plasma hormones were measured in octuplicate in each of six subjects to determine their stability when unseparated from red cells for periods up to 1 week. Most of the analytes were stable when stored in this way and although statistically significant changes were recorded, in the great majority of cases the changes seen would have no bearing on the clinical interpretation of the result. In the light of these findings, we would confidently report results of analyses for these hormones in plasma that had remained in contact with red cells at ambient temperature for long periods of time.

Effects of insulin-like growth factor-I on growth hormone and prolactin secretion and cell proliferation of human somatotrophinomas and prolactinomas in vitro.

OBJECTIVE: IGF-I inhibits GH secretion from normal and some tumorous pituitary tissue, and has been shown to be mitogenic for gonadotrophinoma cells in vitro. It is not known whether IGF-I affects somatotrophinoma cellular proliferation or the secretion of other hormones, such as PRL and alpha-subunit, which are often co-secreted by these tumours. We have therefore examined the effects of IGF-I on proliferation and hormonal secretion of human somatotrophinomas and prolactinomas in vitro. DESIGN: Pituitary adenoma tissue was dispersed to single cells in monolayer culture. The effects of 100 nM IGF-I on GH, PRL and alpha-subunit secretion were determined over 4-hour and over 4-day periods, and a 4-day dose-response study using 1-100 nM IGF-I was performed on two tumours. Adenoma cell S-phase proliferation was determined after bromodeoxyuridine incorporation for 1 hour after 4 days, using a double immunostaining method. RESULTS: Over 4 hours, 100 nM IGF-I had no effect on GH, PRL or alpha-subunit secretion in 7 tumours. Over 4 days, 100 nM IGF-I reduced GH secretion in 5/8 somatotrophinomas (range 17-84%, P < 0.05) compared to controls, with tumours responding to IGF-I having lower basal serum and in-vitro GH levels than tumours unaffected by IGF-I (P < 0.05). There was no effect on alpha-subunit secretion in any of the three tumours studied. PRL cosecretion was increased in 3/5 somatotrophinomas compared to control (20, 30 and 37%, P < 0.05), with tumours responding to IGF-I being associated with lower basal serum and in-vitro PRL levels than those tumours unaffected by IGF-I. IGF-I also increased PRL secretion in 2/2 prolactinomas (27 and 32%, P < 0.05) compared with control. GH was inhibited and PRL secretion was stimulated by 1 and 10 nM IGF-I in the two dose-response studies. The proliferative labelling index did not exceed 1.9% in any tumour and no proliferative effect was found with 100 nM IGF-I in any somatotrophinoma. CONCLUSION: IGF-I inhibited tumorous GH in 62% and stimulated PRL secretion in 71% of tumours over 4 days, without affecting alpha-subunit secretion or being mitogenic for somatotrophinoma cells in vitro. No hormonal effects were observed over short (4-hour) incubations. IGF-I may be a newly recognized factor directly stimulating tumorous PRL secretion.

Differential effects of insulin-like growth factor 1 on the hormonal product and proliferation of glycoprotein-secreting human pituitary adenomas.

The effects of human recombinant insulin-like growth factor 1 (IGF-1) on the secretion, viability, and proliferation of dispersed human anterior pituitary adenomas secreting FSH, LH, and alpha-subunit (alpha-su) were examined in vitro over 4 h and 4 days. The acute effect of IGF-1 on secretion over 4 h was examined in four tumors secreting FSH, LH, and alpha-su. IGF-1 (100 nmol/L) reduced LH compared to control (100%) in one tumor (61%, P < 0.01), and three tumors remained unaffected. FSH and alpha-su secretion were insufficient to measure over 4 h. Nine tumors were studied over 4 days; relative to control, IGF-1 (100 nmol/L) increased FSH secretion in all seven tumors secreting FSH (28-266%, P < 0.05) and increased alpha-su secretion in all four tumors studied (36%, 63%, 91%, and 121%, P < 0.05). IGF-1 reduced LH secretion in four/nine tumors (13%, 23%, 32%, and 50%, P < 0.05). Dose response curves (1-100 nmol/L IGF-1) were performed on three tumors cosecreting FSH and LH. Stimulation of FSH was achieved with either 1 or 10 nmol/L IGF-1, a single tumor in which alpha-su was measured showed maximal stimulation at 10 nmol/L IGF-1, and one of three tumors showed LH inhibition with 100 nmol/L IGF-1. In situ viability of attached cells was assessed with fluorescein and propidium iodide in seven tumors. After 4 days' exposure to 100 nmol/L IGF-1, in situ viability was increased in five tumors (range 12-19%, 15 +/- 1.3% SEM, P < 0.05). The effects of IGF-1 on the adenoma cell proliferative S-phase fraction was determined in six tumors after 4 days of treatment using double immunostaining with bromodeoxyuridine incorporation for 1 h. In four/six adenomas that stained positive for bromodeoxyuridine in the controls (1-5.6%), the S-phase fraction was increased by 100 nmol/L IGF-1 [(range 2.1-10.6%, increase 90-220%) (P < 0.05)]. These results show that IGF-1 has differential effects on gonadotropins from human pituitary adenomas, stimulating intact FSH and alpha-su, inhibiting or being without effect on intact LH in vitro, and increasing both viability and number of tumorous glycoprotein-secreting cells entering into the S-phase of proliferation.

The XY female in sport: the controversy continues.

The summer and winter Olympic Games have been accompanied by much press coverage of the controversy and confusion over sex tests for sportswomen. Much of this has centred on the eligibility of subjects with androgen insensitivity to compete in women's events. The purpose of this paper is to review the process of sex differentiation and its abnormalities, highlighting those conditions in which biologically active testosterone is secreted which might confer an advantage in women's sporting events.

Increased whole body protein breakdown predominates over increased whole body protein synthesis in multiple organ failure.

1. Whole body protein turnover was measured using a primed-constant infusion of L-[1-13C]leucine with measurement of breath 13CO2 production and plasma 13C alpha-ketoisocaproate enrichment. Ten fasting patients, requiring mechanical ventilation and suffering from multiple organ failure, and six healthy control subjects were studied. 2. Protein breakdown and leucine removal from the plasma for protein synthesis were significantly higher in the patients than in the control subjects (P < 0.01). In addition, leucine oxidation was almost 75% higher in the patients than in the healthy control subjects (P < 0.05). 3. Plasma concentrations of glucose, insulin and growth hormone were not different between the two groups, but those of glucagon (not significant), noradrenaline (P < 0.05) and cortisol (P < 0.01) were almost two- and three-fold higher in the patients than in the control subjects. 4. Mean energy expenditure, measured by indirect calorimetry, was 30% higher in the patients than in the healthy control subjects (P < 0.01). 5. Combining the data from both groups of subjects and using multiple regression analysis, cortisol was found to be the most significant predictor of (i) protein breakdown (48% of variance explained), (ii) leucine oxidation (69%) and (iii) hourly energy expenditure (54%). 6. The present investigation using [13C]leucine tracer methods demonstrated, in patients with multiple organ failure, that whole body protein breakdown and synthesis increased concomitantly and were twice as high as rates measured in healthy control subjects. Of the hormones measured in the present study, cortisol appears to have the most significant effect on whole body protein turnover.