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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2751-2759
Copyright © 2002 by The Endocrine Society

Endocrine Care

GH Treatment in Adults with Chronic Liver Disease: A Randomized, Double-Blind, Placebo-Controlled, Cross-Over Study

Jennifer D. Wallace, Winsome J. Abbott-Johnson, Darrell H. G. Crawford, Ross Barnard, Julia M. Potter and Ross C. Cuneo

Metabolic Research Unit (J.D.W., R.C.C.), University of Queensland, Princess Alexandra Hospital, Brisbane 4102; Departments of Dietetics (W.J.A.-J.), Gastroenterology (D.H.G.C.) and Chemical Pathology (J.M.P.), Princess Alexandra Hospital, Brisbane 4102; and Department of Physiology and Pharmacology (R.B.), University of Queensland, Brisbane 4072, Australia

Address all correspondence and requests for reprints to: Jennifer D. Wallace, M.D., Metabolic Research Unit, Department of Medicine, University of Queensland, Princess Alexandra Hospital, Wooloongabba, Brisbane 4102, Australia. E-mail: . jwallace{at}medicine.pa.uq.edu.au

Abstract

Patients with chronic liver disease (CLD) are catabolic and GH-resistant. The effects of supraphysiological recombinant human GH (rhGH; 0.2 IU·kg-1·d-1) treatment in adults with CLD were assessed in a randomized, double-blind, placebo-controlled cross-over trial (4-wk dietary run-in, 4-wk treatment, and 2-wk wash-out phases). Nine adults with mild- to moderate-severity CLD participated (median age, 49 yr; three males and six females; Child’s classification A in six and B in three). Biopsy-proven etiologies were: alcohol (four patients), primary biliary cirrhosis (three patients), non-A, non-B, non-C hepatitis (one patient), and cryptogenic (one patient). Treatment with rhGH increased serum IGF-I (median increase over placebo, +93 µg·liter-1; P = 0.004), IGF-binding protein-3 (+0.9 mg·liter-1: P = 0.004), and acid labile subunit (+10.7 nM; P = 0.004). Total body potassium (+8.0 g; P = 0.023), body weight (+1.6 kg; P = 0.008), and total body water (by bioelectrical impedance; +4.9 kg; P = 0.004) increased. Resting metabolic rate (+313 ml·kg-1·min-1; P = 0.004) and lipid oxidation (+1072.0 kcal·d-1; P = 0.032) increased. Metabolic changes included increased fasting plasma glucose (+1.2 mM; P = 0.008), insulin (+33.8 mU·liter-1; P = 0.004), C-peptide (+0.7 nM; P = 0.004), and free-fatty acids (+0.1 mEq·liter-1; P = 0.04). Clinical side effects included worsening edema and ascites. Hepatocellular function did not change. Therefore, rhGH treatment in CLD: 1) overcame hepatic GH resistance; 2) may have improved whole-body protein catabolism; 3) increased lipolysis and lipid oxidation; 4) increased insulin resistance; and 5) had potent antinatriuretic effects. Long-term safety and efficacy require further assessment.

CHRONIC LIVER DISEASE (CLD), or cirrhosis, is commonly associated with a catabolic state. Reductions of lean tissue and muscle mass are predictive of a diminished survival rate both before and after liver transplantation (1). The etiology of the catabolic state is ill-defined but may include factors such as reduced protein and calorie intake, malabsorption, increased basal metabolic rate (BMR), and abnormalities of intermediary metabolism (2). Nutritional supplementation does little to correct the catabolic state (3).

The GH/IGF axis is deranged in patients with CLD. We have shown that endogenous GH secretion rates in patients with CLD are twice that of normal control subjects, characterized by augmented pulsatile secretion and increased basal concentrations (4). Other studies have shown exaggerated GH responses to a variety of stimuli (5). Worsening hepatocellular function is paralleled by decreased serum IGF-I concentrations. Because the liver is the main source of circulating IGF-I, increased endogenous GH secretion most likely reflects diminished IGF-I-mediated feedback inhibition (4, 6). Therefore, a state of hepatic GH resistance exists in CLD.

The derangement of the GH/IGF axis may contribute to the malnutrition of CLD (2, 4). GH and IGF-I are potent regulators of anabolism in normal adults (7, 8). Low circulating IGF-I concentrations may therefore be important in the genesis of the catabolic state in patients with CLD. Our primary hypothesis was that pharmacological doses of recombinant human GH (rhGH) would overcome hepatic GH resistance, thereby increasing serum IGF-I, and improve the protein catabolic state. In addition, GH stimulates IGF-I production in peripheral tissues, promoting mitogenic and anabolic effects via autocrine and paracrine IGF-I actions (9), so that anabolic effects of rhGH in CLD may also occur independent of changes in circulating IGF-I.

GH has direct lipolytic, insulin antagonistic, and antinatriuretic effects (10, 11). Whether nonhepatic tissues in patients with CLD retain GH sensitivity is unknown. Our secondary hypothesis, therefore, was that endogenous hypersomatotrophism in patients with CLD contributes to metabolic features of cirrhosis (i.e. lipolysis, insulin resistance, and sodium retention), and supraphysiological rhGH treatment may exacerbate these direct, nonhepatic actions of GH.

Materials and Methods

Subjects

The study was approved by the Ethic’s Committees of the Princess Alexandra, Royal Brisbane and Greenslopes Repatriation Hospitals in Brisbane and the University of Queensland. Patients entered after written and informed consent was obtained. The study was conducted in accordance with the Declaration of Helsinki (1964), National Health and Medical Research Council (Australia) Ethics in Medical Research (1983), and Australian Guidelines of Good Clinical Research Practice (1991).

Selection criteria included: 1) age, 18–72 yr; 2) biopsy-proven cirrhosis of the liver; and 3) abstinence from alcohol for at least 6 months if the etiology of the cirrhosis was caused by alcohol. Exclusion criteria included: 1) paracentesis-dependent ascites; 2) significant hepatic encephalopathy, defined as the presence of a metabolic flap, fetor or cerebral dysfunction; 3) serum creatinine over 0.16 mM; 4) congestive heart failure of New York Heart Association class III or IV; 5) hypertension, defined as systolic blood pressure >= 160 mm Hg and/or diastolic blood pressure >= 95 mm Hg; 6) symptomatic hyperglycemia, with random blood glucose consistently more than 10 mM, requiring medication; 7) concurrent chronic infection, e.g. seropositive hepatitis B surface antigen or acquired immunodeficiency syndrome or acquired immunodeficiency syndrome-related complex; 8) treatment with corticosteroid or immunosuppressive drugs; 9) major operation within 1 month; 10) history of a malignant neoplasm other than uncomplicated basal cell carcinoma and squamous cell carcinoma of the skin; 11) gastrectomy; 12) treatment with any form of biosynthetic or pituitary derived GH during the past 3 months; and 13) known hypersensitivity to m-cresol (the preservative used in the treatment medication).

Dose-finding study

To assist in determining an efficacious dose, rhGH (Genotropin; Pharmacia AB, Stockholm, Sweden) was administered, in an unblinded trial, to two individuals fulfilling the selection criteria (a 45-yr-old male with Child’s B alcoholic cirrhosis in abstinence and a 32-yr-old female with Child’s A sclerosing cholangitis) (12). After baseline assessment (0800 h, fasting), consecutive doses of 0.05, 0.1, and 0.2 IU·kg-1·d-1 (17, 33, and 66 µg·kg-1·d-1) were self-administered sc for two nights. Assessments were then made again at 0800 h, fasting, after each dose (i.e. approximately 10 h after the second rhGH dose at each dose level). Results are shown in Table 1Go. No adverse events were noted. The highest dose was chosen for the main study, on the basis of an increase in IGF-I, with the option of the middle dose to retain some efficacy in the event of side effects.


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  		Table 1. Results of a dose-finding study of rhGH treatment

 
Study protocol

Each patient was studied for 14 wk in a randomized, double-blind, placebo-controlled, cross-over study. Treatment consisted of identically presented rhGH (as above) or placebo, which was self administered for 4 wk, as a nightly 2000 h sc injection in the abdominal area, at a dose of 0.2 IU·kg-1·d-1 (66 µg·kg-1·d-1). Patients were seen on seven occasions: an initial screening visit (wk -4), which preceded a 4-wk dietary run-in period, four major study visits (wk 0, 4, 6, and 10) at the beginning and end of the two treatment periods, and on two safety visits in the middle of each treatment phase. A 2-wk washout period separated the treatment periods. The randomization code was computer-generated by Pharmacia AB, Sweden. Randomization was defined at wk 0, after baseline measurements were made. The drugs were administered via the hospital pharmacy. Compliance was assessed by counting returned vials and self-reporting. Blinded dose reduction was permitted for side effects considered possibly to be related to GH therapy.

Procedures: screening visit and 4-wk dietary run-in. A screening visit at wk -4 combined clinical and biochemical screening for eligibility. Patients underwent ventilated-hood indirect calorimetry to habituate them to the procedure before baseline testing and to assist dietary prescription. Patients’ food intake was assessed by an experienced dietitian (W.J.A.-J.), and dietary advice and supplements were prescribed to ensure adequate nutrition. As required, protein intake (adequacy defined as 0.8 g·kg-1·d-1 for ideal body weight; body mass index, 22.5 kg·m-2) was supplemented with liquid Ensure [Abbott Australasia Pty. Ltd. (Health Care), Kurnell, Australia], and total calorie intake (adequacy defined on the basis of measured BMR and estimated physical activity) was supplemented with Polyjoule (Sharpe, Brisbane, Australia). All patients received multivitamins (Elevit RDI; Roche Products Pty. Ltd., Sydney, Australia) for the duration of the study.

Major study visits. Identical procedures were undertaken before and after each treatment period. Patients attended the Metabolic Research Unit, at 0800 h, after an overnight fast. Height and weight were measured in a light cotton hospital gown. Circumferences and skinfold thicknesses were measured by one observer. A baseline urine sample for deuterium oxide (D2O) analysis was obtained after bladder voiding, and a 4-h urine collection for nitrogen excretion was initiated. D2O was administered orally at 0.1 g·kg-1, and a further urine sample for D2O analysis was collected 5 h later. All further measurements were performed with the patient resting supine in bed, in a quiet, air-conditioned room at 23 C. Blood pressure was measured, after 5 min supine rest, by sphygmomanometry and reported as the average of three recordings. Bioelectrical impedance analysis (BIA) was then performed. After a total of 30 min rest (0900 h), BMR and fuel substrate oxidation rates were measured by ventilated-hood indirect calorimetry. At 1000 h, a cannula was placed in an anterior cubital fossa vein for all serum and plasma analytes, as described below, with the patient having remained fasting for approximately 12 h. In addition, serum GH was collected every 20 min, for 2 h (to compare endogenous and exogenous GH levels). After completion of the GH profile, a monoethylglycinexylidide (MEG-X) test was performed to measure hepatocyte function. Patients were examined by a physician for clinical signs of ascites or edema. Analysis of total body potassium (TBK) was performed while still fasting. Dietary intake was reviewed at the end of each visit.

Safety visits. Two weeks after the start of each treatment phase, patients were clinically examined for signs of edema or ascites. Supine blood pressure was monitored; and postprandial, capillary blood glucose concentration was measured by automated glucometer.

Specific test protocols

Body composition. TBK was estimated from the content of naturally occurring radioactive isotope, 40K, measured in a shadow shield, whole-body, thallium-activated sodium iodide crystal counter (Accusan; Canberra Industries, Boston, MA). Correlation between count rate and grams of potassium in a phantom was 0.9995 (13). The coefficient of variation (CV) for repeated measures for this machine in humans was 4% (14). Total body water (TBW) was estimated from isotopic dilution of oral D2O (0.1 g·kg-1 body weight), with urinary D2O measured by mass spectrometry at baseline and after a 5-h equilibration period (15). The in vivo test-retest CV for D2O in adults with CLD was 4.3% (derived from three measurements, over 10 wk, on patients who initially received placebo treatment). TBW and body cell mass (BCM) were estimated by tetra-pole, single-frequency BIA using a SEAC Bioimpedance Meter, model BIM 3.0 (Inderlec, Brisbane, Australia), after bladder emptying. Electrode sites were cleaned with alcohol swabs; and a 350-µA, 50-kHz alternating current was delivered. TBW was calculated according to the equation of Lukaski et al. (16, 17): TBW = 0.377 ht(in cm)2/R+0.14wt (in kg) - 0.08 age (in yr) + 2.9 gender (0 = F, 1 = M) + 4.65 (where R is resistance, at 50 kHz, in ohms). We have previously shown good correlation between TBW estimated from D2O dilution and BIA, in adults with CLD [r = 0.98, n = 19; (15)]. In the current study, correlation between all paired measurements was similar (r = 0.947; P < 0.0001; n = 18). One D2O measurement was a clear outlying value; but because the source of error could not be traced, this result was included in the correlation and outcomes analyses. Skinfold thicknesses were measured by one observer, in triplicate, on the dominant side at the biceps, triceps, subscapular, suprailiac, abdominal, and pectoral sites using a Harpenden skinfold caliper. Circumferences were measured, using a nondistensible tape, at the level of least circumference of the trunk or at the umbilicus (waist), the greater trochanter or maximal gluteal prominence (hip), and the midpoint between the tip of the acromion and the olecranon (midarm). Percentage body fat was calculated from skinfold thicknesses (18).

BMR and substrate oxidation rates. BMR was measured by indirect calorimetry using the Med Graphics CPX-Max Metabolic Cart and ventilated hood system (Medical Graphics, Birmingham, UK). The patient rested supine, with head in an open canopy, in a quiet, dimly lit, thermally-stable room. After 30 min, the canopy was closed. Expired CO2 leaks around the neck air inlet were carefully excluded with adjustments of the air-extraction flow rate. Expired gas was measured for 30 min, with the first 5 minutes discarded from calculations. Gas calibrations were performed immediately before and after each test. The intraclass correlation coefficient of the method was 0.75 (19). Carbohydrate, lipid, and protein oxidation rates were calculated from the O2 uptake and CO2 production rates measured during indirect calorimetry, with a correction applied for urinary nitrogen excretion (20, 21). All urine collected during the 4-h period was acidified to pH 2 with 100% glacial acetic acid, and an aliquot was immediately snap-frozen and stored at -20 C until the batch was assayed using a total nitrogen combustion method (RapidN combustion analyzer; Elementar Analysensysteme GMBH, Hanau, Germany).

Hepatocyte function. Hepatic conversion of an iv bolus of lignocaine (0.1 mg·kg-1) to MEG-X has been shown to be a useful clinical predictor of hepatic function and need for liver transplantation. The metabolite was measured, 15 and 30 min after the dose, as previously reported (22, 23). Childs-Pugh scores were assessed at each major visit, derived from the summed scores of serum bilirubin and albumin, prothrombin time, and the severity of ascites and encephalopathy (24). Liver enzymes were measured by routine methods.

Assays. Plasma samples were collected in chilled tubes and spun immediately. Serum samples were allowed to clot at room temperature for 10 min. Samples were centrifuged at 1000 x g for 10 min in a refrigerated centrifuge, then snap frozen and stored at -20 C. All samples for a given individual, for all visits, were assayed in the same run.

Serum GH was measured by a two-site immunoradiometric assay (Pharmacia AB, Stockholm, Sweden) (4) with between-assay CV of 5.3% at 3.4 mU·liter-1, and 8.7% at 31.9 mU·liter-1. Serum GH-binding protein (GH-BP) was measured by ligand immunofunctional assay (25), with a between-assay CV of 6.4%. Serum IGF-I was measured by RIA after acidified-HPLC (26), with within-assay CV of 3.9 at 316 µg·liter-1 and 1.4% at 418 µg·liter-1, and detection limit of 6.7 µg·liter-1. Serum IGF-II was measured by RIA after acidified-HPLC (27), with within-assay CV of 10.6% at 437 µg·liter-1. Serum IGFBP-3 (28) and acid labile subunit (ALS) (29) were measured using in-house RIAs and polyclonal antibodies. Serum IGFBP-3 within-assay CV were 6.2, 5.5, and 4.5% at 2.5, 5.7, and 12.6 mg·liter-1. Serum ALS within-assay CV were 3.4, 3.3, and 3.4% at 60, 245, and 502 nM. Serum IGFBP-1 was measured by RIA, with within-assay CV of 9.8, 4.6, and 6.4% at 17, 70, and 250 µg·liter-1 (Diagnostic Systems Laboratories, Inc., Webster, Texas). Other assays included: plasma insulin (in-house RIA; intraassay CV <5%), C-peptide (Diagnostic Systems Laboratories, Inc.; RIA; intraassay <7.8%), glucagon (Diagnostic Products Corp., Los Angeles, CA; RIA intraassay CV <15.7%), serum free T4 (competitive immunoassay, Ciba Corning, Inc., Medfield, MA; intraassay CV <5.2%), free T3 (Amerlex-M free T3 RIA kit, Int. PLC, Amersham Pharmacia Biotech, Amersham, UK; intraassay CV <10%), FFA (enzymatic colorimetric assay, NEFA-C kit, Wako Pure Chemical Industries Ltd., Osaka, Japan; intraassay CV at 0.99 mEq·liter-1 = 1.1%), and ß-hydroxybutyrate (in-house enzymatic spectrophotometric assay and COBAS FARA II analyzer, Roche Pharmaceuticals, Zurich, Switzerland; interassay CV 6.6% at 0.61 mM). Plasma amino acids were measured by ion-exchange HPLC and ninhydrin postcolumn spectrophotometry (interassay CV, 13.6% and 7.7% for glutamine and glutamate, respectively).

Nutritional assessment: diet and grip strength. Dietary recall, over the preceding 24 h, was analyzed by an experienced dietitian using the Diet/1 software program (Xyris Software Pty. Ltd., Brisbane, Australia) based on Nuttlab 95 food database (Australian and New Zealand Food Authority, Canberra, Australia). Bilateral grip strength was measured with a Smedley dynamometer (VacuMed, Ventura, CA); and the average of the two sides was reported (30).

Statistical analysis

The treatment code was not broken until all patients had completed the study and clean file status of the database was verified. Final statistical analysis was performed by Instat, Canberra, Australia, independent of the investigators and sponsor. Because of the small data set and nonnormal distribution of some endpoints (assessed by the Shapiro-Wilk test), all data were analyzed nonparametrically and results presented as median and range (25th and 75th percentiles). Order, period, carry-over, and treatment effects were assessed by the Wilcoxon signed-rank test. Carry-over effects were defined as the change in variable between the start of treatment phases 1 and 2, and they were assessed for primary endpoints only, by comparing those receiving rhGH or placebo first with the Mann-Whitney independent-rank sum test. Prior power calculations predicted 20 patients would be required for significant changes in the three primary endpoints (IGF-I, TBK, and BMR) with 90% probability. Blinded interim analysis was planned after 8 and 15 completed patients; the trial was stopped after the first analysis because changes in all primary endpoints were significant. Statistical significance was ascribed at P < 0.05. Simple linear and multiple regression analysis was used to assess relationships between GH-BP and IGF-I.

Results

Patient characteristics are presented in Table 2Go. Before treatment, one Child-Pugh class A and all class B patients received diuretics (frusemide, n = 3; spironolactone, n = 4; or amiloride, n = 1). One patient had nonparacentesis-dependent ascites. Other medications included: treatment for reflux or gastropathy (omeprazole, n = 1; ranitidine, n = 2; cisapride, n = 1), encephalopathy (lactulose, n = 2), portal hypertension (propranolol, n = 1; isosorbide dinitrate, n = 1), pruritus (methdilazine, n = 1), pain (dextropropoxyphene/paracetamol, n = 1; paracetamol, n = 1; codeine, n = 1), constipation (psyllium, n = 1; bisacodyl, n = 2), asthma (salbutamol, n = 1), renal calculi (sodium bicarbonate, n = 1), and combination estrogen/progesterone replacement (n = 1). Eleven patients entered the study; two patients withdrew before randomization (one protocol violation for alcohol intake and one consent withdrawal). Of the nine randomized, one patient completed the study to the end of the first treatment phase (placebo) and was withdrawn because of surgical resection of an ovarian cyst; she subsequently reentered, was rerandomized, and completed the entire protocol. Therefore, nine patients completed the entire protocol. Treatment compliance approached 100% (one patient missed one injection). There were no order or period effects in any measure, except for an order effect for eosinophil count and FFA (each P = 0.03). There were no carry-over effects detectable for serum IGF-I (P = 0.18), TBK (P = 0.54), or BMR (P = 0.33).


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  		Table 2. Baseline patient characteristics (median and 25th, 75th percentiles)

 
Changes in the GH/IGF axis are presented in Table 3Go, with selected individual data in Fig. 1Go. Serum IGF-I at baseline was below the age-specific reference value in 6 out of 9 patients (ages 19–60 yr, 75–376 µg·liter-1 (for males); ages 19–55 yr, 75–414 µg·liter-1, and ages >55 yr, 60–226 µg·liter-1 (for women), based on 384 normal South Australian adults; unpublished data). There was a strong relationship between pretreatment serum GH-BP and serum IGF-I (Fig. 2Go). In response to treatment, serum IGF-I almost trebled, normalizing in 8 and increasing outside the normal range in 1 patient. Pretreatment serum GH-BP was very low in most patients before treatment (normal for males, 0.50 ± 0.04; and females, 0.80 ± 0.13 nM), remained unchanged, but did not predict the IGF-I response to treatment (data not shown). Serum IGFBP-3 at baseline was low in 7 out of 9 patients (normal, >2.2 mg·liter-1), doubled with treatment, normalizing in 3 of those 7 and increasing outside the normal range in one. Serum ALS also doubled. Those patients with the better clinical status seemed to respond with greater increments (see Fig. 1Go). Fasting serum IGFBP-1 concentrations were normal before treatment (<150 µg·liter-1) and decreased to less than half of baseline concentrations after treatment.


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  		Table 3. Changes in the GH/IGF axis after rhGH or placebo treatment

 


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  		Figure 1. Changes in the rhGH and placebo phases of treatment in adults with CLD. Patients with Child-Pugh classification A (solid symbols and lines) and B (open symbols and dotted lines) are shown separately.

 


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  		Figure 2. Relationship between pretreatment serum GH-BP and serum IGF-I in adults with CLD.

 
Body composition data are presented in Table 4Go. TBK was low at baseline, with a median baseline value of 88.4% of predicted value, (range, 66.5–103.4%). GH treatment significantly increased TBK, by a median of 10.5%. BCM, estimated from BIA, increased 10.4%. Skinfold thicknesses did not change (data not shown). TBW increased after GH treatment, by 18.1%, as assessed by BIA (P = 0.002; n = 9). Several missing data points and one outlying value may have precluded a similar conclusion regarding TBW, assessed by deuterium dilution (+7.1%; P = 0.4, n = 7). Clinically, worsening ascites and edema were side effects in four patients. Blinded halving of the rhGH dose, caused by side effects during the active phase of treatment, was required in six out of the nine patients. One patient was able to resume the prescribed dose after spontaneous reduction in edema. Median grip strength tended to decrease after rhGH treatment (median, -2.5 kg; 25th and 75th percentiles, -10 and -1.5 kg; n = 9; P = 0.04). There were no changes in reported carbohydrate or protein intake.


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  		Table 4. Body composition changes after rhGH or placebo treatment

 
Metabolic rate and fuel oxidation rates are presented in Table 5Go. BMR before treatment was elevated, with a median value of 116% that of predicted normal. Before treatment, fuel oxidation rates and the RQ (ratio of CO2 production to O2 consumption) of 0.7 indicated lipid as the predominant substrate for fuel oxidation. After rhGH treatment, BMR increased by 19.4%. This thermogenic response was accompanied by a further increase in lipid oxidation, a trend toward reduced carbohydrate oxidation, and no change in protein oxidation.


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  		Table 5. Indirect calorimetry and fuel oxidation changes after rhGH or placebo treatment

 
Metabolic data are presented in Table 6Go. Fasting plasma glucose increased modestly, with the highest concentration being 6.7 mM after rhGH. Plasma insulin was modestly elevated before treatment (normal, <10 mU·liter-1) and quadrupled after treatment. Plasma C-peptide was similarly elevated before treatment (normal, 0.26–0.70 nM) and increased moderately with treatment. Plasma FFA were elevated at baseline (normal, 0.06–0.75 mEq·liter-1) and tended to increase further with GH treatment. Serum ß-hydroxybutyrate was normal before treatment (<0.18 mM) and trended upward with treatment. Serum glutamate was elevated before treatment (10–50 µM) and decreased with treatment. Serum glutamine was normal before treatment (420–700 µM) and tended to increase with treatment. Alanine was normal before treatment and remained unchanged. Aromatic amino acids were normal before treatment and did not change with treatment (pretreatment phenylalanine median, 62; range, 51–72; normal, 40–120 µM) (tyrosine median, 88; range, 74–95; normal, 50–90 µM). Serum total albumin-corrected calcium and phosphate increased. Serum urea did not change. Thyroid function was normal before treatment; free T4 decreased, but free T3 did not change after treatment.


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  		Table 6. Metabolic changes after rhGH or placebo treatment

 
Safety assessment revealed one patient changed from Child-Pugh class B to A during rhGH treatment (i.e. an improvement), whereas no change was noted during placebo treatment. There were no significant changes in bilirubin, prothrombin, or albumin (downward trend; P = 0.06). Serum liver enzymes remained stable (lactate dehydrogenase, aspartate amino transferase, and alanine amino transferase) or trended downwards ({gamma}-glutamyl transpeptidase, P = 0.05; alkaline phosphatase median, -43 U·liter-1, P = 0.004). Lignocaine clearance remained unchanged. Fasting serum glucose increased (Table 6Go), and all concentrations were within normal limits; one patient developed transient postprandial hyperglycemia (capillary glucose, 16.8 mM) with subsequent normalization. Blood pressure and serum creatinine did not change.

Discussion

The key findings of this study were that supraphysiological rhGH treatment in adults with CLD: 1) overcame preexisting GH resistance, as shown by the increase in serum concentration of the GH-dependent proteins, IGF-I, IGFBP-3, and ALS; 2) induced a possible anabolic benefit; 3) induced a definite thermogenic response, with increased lipolysis and lipid oxidation; 4) increased insulin resistance; and 5) had potent antinatriuretic effects.

Patients with CLD have low serum IGF-I and related IGF-binding protein (IGFBP) concentrations and elevated GH secretion rates (4). This state of hepatic GH resistance is consistent with reports of absent or reduced GH receptors in cirrhotic liver (31, 32). Our hypothesis was that supraphysiological doses of rhGH could overcome the hepatic GH resistance and increase IGF-related protein concentrations. Serum GH concentrations, 11–13 h after the evening sc rhGH administration, were not different from pretreatment, endogenous levels, suggesting that the peak of absorption had passed (33). Serum IGF-I increased (in most cases, into the normal range). Our data extend the observations of previous workers (34, 35) by showing similar increments in serum IGFBP-3 and ALS. Hepatic Kupfer cells produce IGF-I, and hepatocytes produce IGFBP-3 and ALS (36, 37). GH strongly regulates synthesis of IGF-I and ALS and (to a lesser extent) IGFBP-3 (38). Whereas nonhepatic tissues synthesize IGF-I and IGFBP-3, ALS secretion seems to be restricted to the liver (37). These data suggest that the parallel increments in these components of the IGF-I ternary complex were of hepatic origin, but the exact source remains to be established. The greater IGF-related increments in patients with less severe liver dysfunction suggest that the reduction in hepatic tissue GH receptor status paralleled clinical worsening of cirrhosis (39).

GH-BP in humans is a circulating polypeptide with an amino acid sequence identical to that of the extracellular portion of the human GH-receptor (40), derived as a cleavage product of the mature receptor. It has been proposed that circulating GH-BP concentrations may reflect tissue GH-receptor density (41). In this study, pretreatment GH-BP concentrations were low and strongly associated with pretreatment serum IGF-I, supporting this hypothesis (42). In response to rhGH treatment, serum GH-BP did not change, similar to other clinical situations (43). Whether pretreatment serum GH-BP (and, by inference, tissue GH receptor status) predicts the IGF-I response to rhGH treatment in CLD remains to be established, given that our data set was probably too small to test this hypothesis.

Was rhGH treatment anabolic? In this study, GH treatment resulted in an increase in lean tissue mass or BCM of approximately 10%, assessed by two independent analyses (TBK and bioelectrical impedance). We are cautious, however, in accepting these data without additional validation. Potassium is the main intracellular ion, and TBK is a reliable measure of BCM or lean body mass in normal and obese individuals (44). Alterations in intracellular potassium concentration and extracellular vs. intracellular water distribution, after rhGH treatment in patients with CLD, may reduce the precision of TBK and bioelectrical impedance, respectively, to measure lean body mass (45). GH has been shown to activate the Na/K ATPase pump (46), potentially increasing intracellular potassium independent of a true increase in BCM. On the other hand, comparable increases in TBK (+8%) and muscle mass (+5%) have been shown after rhGH treatment in adults with GH deficiency (47). The small reduction in grip strength in the current study almost certainly reflected fluid retention and/or arthralgias, known effects of supraphysiological rhGH treatment (48). Studies of longer duration and using additional techniques will be required to further assess body composition and function.

GH has been shown to increase amino acid uptake, and to increase protein synthetic rates in whole body or limb perfusion studies in humans (49). We documented an increase in serum glutamine after rhGH treatment, the significance of which is unknown. Glutamine is an important component of the catabolic response to critical illness (50), but whether the changes observed in the current study reflected an increase in the mobilizable pool available for protein synthesis or a reduction in utilization requires further study. Serum glutamate decreased after rhGH treatment. Glutamate is formed from glutamine by glutaminase and is destined to provide alanine for gluconeogenesis (51, 52). One possible explanation for the changes in glutamine and glutamate after rhGH treatment may therefore be a reduced requirement for gluconeogenesis. Glutamate is also a GH-secretagogue (53), prompting the speculation that the high pretreatment glutamate concentration contributed to the prevailing hypersomatotrophism.

Treatment with rhGH clearly had a thermogenic effect. Our patients had pretreatment BMR 16% higher than predicted from anthropometric data, consistent with previous reports (54), and rhGH treatment resulted in a further 19% increase. Similar responses to rhGH treatment are seen in other conditions (8, 55). The mechanism for the rhGH-mediated increase in BMR in cirrhosis may relate to alterations in fuel utilization and/or body composition. Before treatment, serum FFA concentrations were elevated, and the RQ was 0.7, indicating enhanced lipolysis and a predominance of lipid oxidation (21). After rhGH, we observed a further increase in serum FFA, which presumably reflected GH’s acute lipolytic effect. In addition, lipid oxidation increased dramatically, with no significant changes in carbohydrate or protein oxidation. Energy-consuming processes, such as substrate cycling, were not assessed. An increase in BCM would be energetically demanding because of the generation and maintenance of the increased mass of metabolically active tissue. A GH-mediated increase in increased Na/K ATPase pump activity may also have occurred (46, 56). Finally, rhGH-induced increased conversion of T4 to T3, which is thought to explain some of the thermogenic effects of rhGH in GH-deficient adults (57), was not seen in our cirrhotic patients. The combination of reduced serum T4 and unchanged serum T3 seen in the current study may reflect reduced TSH-mediated thyroid hormone production combined with accelerated GH-mediated T4-to-T3 conversion, or preferential conversion of T4 to reverse T3.

Cirrhotic patients are insulin resistant (58). As assessed by normal fasting plasma glucose and increased plasma insulin and C-peptide concentrations, our patients had a degree of insulin resistance before treatment. Insulin resistance increased markedly after rhGH treatment. One patient developed transient hyperglycemia; future studies will need to carefully assess this aspect. Also, another patient, with relatively frequent symptoms of fasting hypoglycemia and fasting serum glucose of 2.7 mM, was symptom-free and normoglycemic while on rhGH treatment. Hyperinsulinemia may have been beneficial in these patients: first, by causing a decrease in serum IGFBP-1, which potentially increased free IGF-I (59), thereby contributing to anabolism; second, hyperinsulinemia may itself represent a direct anabolic stimulus (49). We also speculate that GH’s antagonism of insulin’s action in cirrhosis extends beyond carbohydrate to lipid metabolism, given the predominance of lipid as a source of fuel oxidation. Also, we noted a small ketogenic effect of GH (60), again suggesting rhGH-induced insulin resistance in terms of carbohydrate and lipid metabolism.

Patients with CLD commonly develop osteoporosis or osteomalacia. GH treatment resulted in small (but significant) increases in serum calcium and phosphorus concentrations, possibly reflecting vitamin D-mediated effects, direct renal effects, and/or an increase in bone turnover (61).

The other main effect of GH treatment noted in this study was the dramatic antinatriuretic action. Assessment of TBW by BIA and D2O yielded comparable results. D2O is generally regarded as the gold standard measure of TBW, but this result did not reach statistical significance, as a result of missing and outlying data points. We report the results without deleting the statistical outlying value so as not to introduce bias. Clinically, some patients gained 8 kg body weight in 2 wk, along with signs of edema and ascites, effects which only partly regressed on a lower GH dose. Whereas GH’s antinatriuretic action is well recognized (62), the magnitude of changes in patients with CLD was surprising, probably reflecting the combination of the preexisting secondary hyperaldosteronism in CLD and GH’s antinatriuretic action. Our data clearly suggest that GH-mediated antinatriuretic mechanisms remain operative in patients with CLD. Therefore, we speculate that, given the known hypersomatotrophism of CLD (4), endogenous GH excess may contribute an additional mechanism favoring sodium retention in such patients.

GH treatment seemed to be safe, with no changes in hepatocellular function, as assessed by the MEG-X test, serum bilirubin and albumin, and prothrombin times. Reductions in alkaline phosphatase and {gamma}-glutamyltranspeptidase suggested that biliary inflammation may even have improved. Also, there were no changes in aromatic amino acids. Given that these have been implicated in the pathogenesis of hepatic encephalopathy, it is encouraging to note that there were no clinical signs of hepatic encephalopathy in any patient during the study.

There are several limitations of our study. The rhGH dose was chosen on the basis of a pilot study using only 2 d treatment at each dose. When the study was designed, we were unsure whether serum IGF-I would prove responsive. The final dose chosen on the basis of some effect on IGF-I and BMR may have been excessive, because 4–7 d are needed for a steady-state serum IGF-I response. Though the dose was similar to other catabolic illness studies at the time and appeared efficacious as assessed by the serum IGF-I achieved, the delayed-onset sodium and fluid retention could not have been anticipated from the pilot study. Our results should encourage the use of lower rhGH treatment doses in future studies. The possibility that the study became unblinded because of these side effects must be considered. We suggest, however, that the overall results of this study are valid, because several of the patients were accustomed to intermittent changes in edema, we did not speculate on treatment phase allocation with the patients during the trial, and all end-points apart from grip strength were objective and not effort-dependent in nature.

In summary, rhGH treatment in CLD can overcome GH resistance and increase serum IGF-I. Thermogenic and lipolytic responses, comparable with those in trials of rhGH in anabolic-support conditions, were seen. Whether a substantial anabolic improvement in body composition also occurs in patients with CLD, whether such effects can be realized without excessive sodium retention, and at what dose of rhGH, remain to be determined.

Acknowledgments

We are indebted to the patients and their referring clinicians (Drs. Paul Kerlin, Charles Steadman, Steven Lynch, Russell Strong, Andrew Hallam, and Graham Stevenson); to Tony Dique and Karen Peacock for excellent clinical assistance; to Greg Ward and Drs. Francis Thomas, Joan Skinner, and Alan Clague (of Queensland Health) for biochemical assistance; to Prof. Robert Baxter (Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney) for IGF-BP assays and review of the manuscript; to Dr. Peter O’Leary (Department of Biochemistry, King Edward Memorial Hospital, Perth) for IGFBP-1 assay; to Phil Owens (Cooperative Research Center for Tissue Growth and Repair, Adelaide) for IGF assays; to Paul Masci for technical assistance; to Michael Jones (Instat Australia Pty. Ltd.) and Diana Battistuta (Medical Biostatistics Pty. Ltd.) for statistical assistance; to Sue Golding for the D2O assays; to Dr. Ross Shephard and technical staff for the TBK scanning; to Peter Martin (Queensland Department of Primary Industries) for urinary nitrogen analysis; and to Sharon Hammond and Phillipa Smith (from Pharmacia Australia Pty. Limited) for patience and professionalism.

Footnotes

This work was supported by grants from the Princess Alexandra Hospital Research and Development Foundation and Pharmacia Australia Pty. Limited. Part of this work was presented at The International Congress of Endocrinology meeting, San Francisco, California, 1996.

Abbreviations: ALS, Acid labile subunit; BCM, body cell mass; BIA, bioelectrical impedance analysis; BMR, basal metabolic rate; CLD, chronic liver disease; CV, coefficient of variation; D2O, deuterium oxide; GH-BP, GH-binding protein; IGFBP, IGF-binding protein; MEG-X, monoethylglycinexylidide; rhGH, recombinant human GH; RQ, respiratory quotient; TBK, total body potassium; TBW, total body water.

Received May 4, 2001.

Accepted February 15, 2002.

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