Unresponsiveness of hepatic nitrogen metabolism to glucagon infusion in patients with cirrhosis: Dependence on liver cell failure

Unresponsiveness of Hepatic Nitrogen Metabolism to Glucagon Infusion in Patients with Cirrhosis: Dependence on

Liver Cell Failure

ANDREA FABBRI, ' GIULIO MARCHESINI, GIAMPAOLO BIANCHI,' ELISABETTA BUGIANESI, LUCIA BORTOLUZZI,' MARC0 ZOLI' AND EMILIO PISI'

'lstituto di Clinica Medica Generale e Terapia Medica, University of Bologna, and 2Laboratorio Centralizzato, Policlinico S. Orsola, I-40138 Bologna, Italy

Glucagon exerts an up-regulatory effect on hepatic nitrogen metabolism in healthy subjects, but its potential role in the presence of liver failure is uncertain. The effects of glucagon on urea synthesis and hepatic nitrogen clearance during alanine infusion were studied in five control subjects and six cirrhotic patients in paired experiments at spontaneous glucagon concentrations and at high physiological glucagon levels (=300 to 500 pmol.L-') induced by a 7.5-hr continuous glucagon infusion. In all experiments the urea nitrogen synthesis rate increased linearly with increasing a-amino-nitrogen concentrations. At spontaneous glucagon concentrations the dynamics of a-amino nitrogen to urea nitrogen conversion (functional hepatic nitrogen clearance) were significantly reduced in cirrhosis (23.2 f 6.7 L . hr- vs. 35.3 f 8.0 Lshr-', p < 0.05) in relation to decreased liver function. Glucagon superinfusion caused a 63% increase in the dynamics of the process in controls (57.7 -+ 11.0 La hr-'; p vs. spontaneous glucagon, p < 0.01), whereas in cirrhosis it increased on average by only 15% (26.7 -C 10.7; p 1 NS). The glucagon-induced change in functional hepatic nitrogen clearance significantly correlated with galactose elimination capacity and antipyrine clearance (r = 0.905 and 0.964, respectively). Glucagon, in high physiological concentrations achieved with glucagon infusion, does not produce significant effects on hepatic nitrogen metabolism in cirrhosis. The reduced sensitivity of the cirrhotic liver to glucagon seems to be dependent on decreased hepatocellular function. These data do not support the role of glucagon as a "catabolic" hormone in cirrhosis. (HEPATOLOGY 1993; 18:28-35.)

The liver is the target organ for the up-regulatory effect of glucagon on protein and amino-acid metab-

Receivrd June 12, 1992; accepted Januarj 26, 1993. This work was supported by a grant from Minister0 dell'Universita e della

Ricerca Scientifica e Tecnologica (MURSTi. Rome, Italy, Fondi 60%,, 1990. Address reprint requests to: Giulio Marchesini, M.D., Istituto di Clinica

Medica Generale e Terapia, University of Bologna, Policlinico S. Orsola, Via Massarenti 9, 1-40138 Bologna, Italy.

Copyright ' x ' 1993 hy the American Association for the Study of Liver Diseases

0270-9139193 $1.00 + .10 3111146555

olism. Glucagon has been shown to increase amino-acid transport in the liver (1) and to induce the enzymes of the urea cycle (21, resulting in increased urea nitrogen synthesis rate (UNSR). By regulating plasma amino-acid concentrations through their disposal in the liver and the rate of gluconeogenesis (31, glucagon may also contribute to amino-acid/protein metabolism in peripheral muscle tissue and to whole-body nitrogen economy.

The capacity of the liver to convert amino-acid nitrogen into urea nitrogen at any a-amino nitrogen concentration is regulated by glucagon (4, 3, and in the physiological range there is a trend toward dose dependence ( 5 ) . However, the dynamics of the process also depend on functional liver cell mass (61, as shown in experimental animals after graded hepatectomy ( 7) and in patients with acute (8) and chronic (9) liver failure.

Elevated glucagon levels have been described in patients with acute (8, 10) and chronic (11, 12) liver disease. In patients with cirrhosis glucagon seems ineffective in stimulating gluconeogenesis (13, 141, but no data are available on the possible stimulatory effect of glucagon on hepatic nitrogen metabolism.

In an attempt to study the potential role of glucagon in the regulation of hepatic nitrogen kinetics, we measured the effects of glucagon infusion, to attain high physiological concentrations, on a-amino nitrogen into urea nitrogen conversion in control subjects and in patients with cirrhosis. The results show that extra glucagon has little or no effect on the dynamics of hepatic nitrogen metabolism in cirrhosis in relation to the extent of liver failure.

MATERIALS AND METHODS Subjects. Five male control subjects and six male patients

with cirrhosis were studied. Patients, aged 42 to 57 yr (median = 51 yr), had cirrhosis of alcoholic origin (three cases), hepatitis C virus antibody (two cases) and HBV (one case). Patients with alcoholic cirrhosis had been abstaining from alcohol for at least 6 mo. Liver function parameters are reported in Table 1. Two subjects were in fairly good condition, without nutritional defects, whereas two had clinical evidence of reduced lean body mass. Child-Pugh class ( 15) was A in two cases, B in one case and C in the remaining three cases. Five

28

HEPATOLOGY Vol. 18, No. 1, 1993 FABBRIETAL. 29

patients had esophageal varices (small in one case, medium- sized in two and large in two). One had been repeatedly treated by endoscopic sclerotherapy for recurrent gastrointestinal bleeding. Three had ultrasonographic but not clinical evidence of ascites at the time of study; five were under diuretic treatment with spironolactone, furosemide or both. Signs of hepatic encephalopathy had been previously demonstrated in three patients, who were being treated with lactulose.

Control subjects (39 to 62 yr; median = 47 yr) had been hospitalized for mild gastrointestinal symptoms. Liver function parameters were in the normal range, and patients had no evidence of previous or actual liver disease.

All subjects had normal kidney function (creatinine < 1.2 mg . dl ~ '). Before the study, patients consumed a standard hospital diet to provide 30 to 35 kcal and more than 1 gm proteinkg body wt for at least 5 days. In all cases the galactose elimination capacity was measured according to Tygstrup's technique ( 161, whereas antipyrine clearance was measured with a two-sample procedure (17).

All subjects gave their informed consent. Because we have no ethical committee in our department, the protocol was submitted to and approved by the senior staff committee.

Methods. Urea synthesis was studied in relation to intra- venous alanine infusion, as previously described (18), during paired experiments (with and without exogenous glucagon) performed at 7- to 10-day intervals, in random order. Subjects did not eat during the test.

In the basal experiment (at spontaneous glucagon concentrations), only alanine (Ajinomoto Co. Inc., Tokyo, Japan; 10% wtlvol, water solution) was infused, at a constant rate of about 2 m r n 0 1 ~ k g ~ " ~ ' - ' , f or 4.5 hr. Blood samples were obtained from a vein of the contralateral arm every 45 min during alanine infusion and 90 min before and 90 min after the infusion. Throughout the experimental period, urine was collected quantitatively by voiding in five consecutive 90-min periods.

The experiment with exogenous glucagon was basically the same, except that glucagon (NOVO Industri, Copenhagen, Denmark) was infused throughout the experimental period at a constant rate of = 300 pmol * kg- 1. hr - in isotonic solution containing 20% human albumin (2.5% volivol). The concentration of glucagon in the infusate was calculated to yield plasma glucagon concentrations of = 300 to 500 pmol - L (i.e., three to 10 times normal levels). Such values may occasionally be measured in patients with cirrhosis.

Both infusions were given with calibrated roller pumps (Infusomat Secura; Braun, Melsungen, Germany).

In both experiments urine flow was stimulated by peroral water or saline solution infusion to keep diuresis greater than 2 ml . min- '. This was achieved in nearly all subjects (mean diuresis = 2.2 ml * min- I ) , but diuresis was greater in controls (2.8 ? 0.8 ml-min- ') than in subjects with cirrhosis (1.8 t 0.8 mlemin-I). The amount of water given in paired experiments was the same. No side effects or complications to the infusion of alanine were noted. In particular, no subjects complained of nausea or vomited, and no patients with cirrhosis exhibited signs of hepatic encephalopathy.

The urea nitrogen synthesis rate during each time period was measured as the sum of the urea nitrogen excretion rate in urine and the accumulation of urea nitrogen in the urea space, assumed to equal total body water (TBW), as

UNSR = (E + A)/(l - L)

where E = (Urine fiow [L * hr- 'I) - (Urinary urea nitrogen [mmol * L- l]); A = (Change in blood urea nitrogen

[mmol . L - * hr-l]) * (TBW [L]); and L = (Fractional loss of newly formed urea in the gut).

TBW was calculated on the basis of antipyrine distribution space (19). It was similar in the two groups (Table 1) and accounted for 60.2 2 4.0% of total body weight in controls and for 65.4% -C 8.7% in cirrhosis. Intestinal loss of urea nitrogen due to bacterial hydrolysis was taken to be 0.17 in controls and 0.26 in patients with cirrhosis (20).

Functional hepatic nitrogen clearance (FHNC) was calculated as the slope of the linear regression analysis of UNSR on average a-amino nitrogen concentration during each time period (mean of a-amino nitrogen values measured at the beginning and at the end of each urine collection).

Laboratory Procedures. Urea nitrogen concentrations in plasma and urine were measured by the urease Berthelot method (21). Alanine was measured enzymatically (221, and total ci-amino nitrogen was assayed by the dinitrofluo- robenzene method (23). All analyses were carried out in batches, in duplicate or triplicate, to minimize analytical error. In our laboratory the intraassay coefficients of variation are urea, 5 1.5%; a-amino nitrogen, ?2%; and alanine, ?3%. Plasma amino-acid concentration profile was measured by ninhydrin reaction after ion-exchange chromatography at baseline and at the end of alanine infusion (24). Repeated analyses of plasma amino acids gave a coefficient of variation less than 5%. Plasma insulin and glucagon were measured by means of insulin and glucagon kits (Biodata-Serono, Guidonia, Italy). The coefficients of variation for insulin and glucagon were, respectively, 8% and 11% (intraassay) and 12%' and 14% (interassay). Glucose was measured enzymatically.

Statistical Analysis. Linear correlation analysis between variables was performed with the least-squares method (25). Differences between mean values of groups were tested with Student's t test and one-way ANOVA. Differences between paired data were analyzed by paired Student's t test. With respect to changes in amino-acid concentration during alanine infusion, we performed multiple simultaneous comparison. Therefore, to avoid the risk of mass significance, the significance level was adjusted according to Duncan's multiple range (26) by the formula

where n is the number of measured amino acids (threonine, serine, glutamine, proline, glycine, alanine, methionine, sum of branched-chain amino acids [valine, isoleucine and leucinel, sum of aromatic amino acids [phenylalanine and tyrosinel, ornithine, lysine, histidine, arginine) and p = 0.05. Data in the text and in the tables are given as mean 2 S.D.

RESULTS Spontaneous Glucagon. In the fasting preinfusion

state, blood glucose level was significantly higher in cirrhosis, but no patients had values exceeding 8 mmol * L- '. Also, plasma glucagon and insulin levels were higher (by nearly 100% and 50%, respectively) (Table 2). No differences were found in a-amino nitrogen levels. Alanine infusion nearly doubled glucagon level in both groups, whereas insulin level did not change significantly. &-Amino nitrogen levels peaked at an average concentration greater than 7 mmol * L ~ l.

Basal UNSR was similar in the two groups (20 5 10 mmol - hr in controls and 19 L 13 nmol- hr- l in cirrhosis) and increased several times with increasing

30 FABRRIETAL. HEPATOLOCY July 1993

TABLE 1. Clinical and laboratory data in patients with cirrhosis and control subjects

Age Weight TBW Albumin Case no. Pathogenesis of cirrhosis (Yr) tka) (L) ( m . L - ' )

Cirrhotics 1 2 3 4 5 6

Mean f S.D. Controls Mean t S.D. Range

Hepatitis C Alcohol Alcohol Alcohol Hepatitis C Hepatitis B

42 47 57 51 51 53

50 2 5

49 2 9 39-62

75 70 73 76 80 51

70.8 t 9.4

73.6 t 7.4 65-87

49.9 39.6 49.3 43.4 49.6 42.1

45.0 f 3.9

44.1 t 3.6 37.9-47.9

37.3 38.0 29.9 39.8 41.7 31.3

36.3 t 4.7

39.9 t 2.6 35.9-42.5

TABLE 2. Glucose, insulin, glucagon, ammonia and a-amino nitrogen concentrations at the beginning and end of alanine infusion in the course of paired experiments

Experiment Before alanine infusion End of alanine infusion p Value

Spontaneous glucagon Glucose Control 5.1 t 0.8" 5.0 t 0.7 NS

Cirrhotic 5.6 c 1.1* 5.4 t 0.9 NS Insulin Control 75 L 38 68 rt 31 NS

Cirrhotic 110 ? 50 163 t 87 < 0.05 Glucagon Control 30 2 6 55 t 17 < 0.01

Cirrhotic 72 2 36* 145 f 24* <0.001 Ammonia Control 17 ? 3 30 t 6 < 0.01

Cirrhotic 27 -c 8 58 i 11 < 0.01 a-Amino nitrogen Control 3.5 c 0.8 7.5 2 2.3 < 0.01

Cirrhotic 2.7 2 0.4 7.5 t 2.9 < 0.01

Glucose Control 6.2 2 0.8' 5.2 0.9 <0.01 Cirrhotic 6.4 ? 0.8 5.3 2 1.2 <0.01

Insulin Control 131 i- 50 149 f 52 NS Cirrhotic 177 2 66 153 ? 49 NS

Glucagon Control 190 f 82" 400 f 240" <0.001 Cirrhotic 268 ? 161' 368 +- 179' < 0.001

Ammonia Control 14 2 4 22 t 6 < 0.01 Cirrhotic 28 ? 7 58 +- 11 < 0.01

a-Amino nitrogen Control 3.4 2 0.9 6.1 2 1.2" < 0.01 Cirrhotic 2.7 ? 0.4 7.1 f 2.2 < 0.01

Glucagon superinfusion

Glucose and a-amino nitrogen levels are expressed as mmol . L- I; ammonia is expressed as kmol. L- '; glucagon and insulin are expressed

"Data expressed as mean 2 S.D. *Significantly different from the corresponding control value. 'Significantly different from the corresponding value in the experiment carried out a t spontaneous glucagon concentrations.

as pmol . I, ~ '.

a-amino nitrogen concentrations. During the last period of alanine infusion, UNSR was 23% lower in cirrhosis (126 ? 31 mmol hr- vs. 164 ? 28 nmol - hr- l ; 0.1 > p > 0.05). The relationship of UNSR to the average a-amino nitrogen in each time period was linear in the experimental range and in both groups (r = 0.80 to 0.98).

FHNC varied within and between the two groups (Fig. 1); it was reduced, on average, by 35% in cirrhosis (23.2 2 6.7 L * hr- vs. 35.3 * 8.0 L * hr- ' in controls; p < 0.025). In the whole group, FHNC correlated significantly with galactose elimination capacity and antipyrine clearance (r = 0.852 and r = 0.812, re-

respectively; n = 11; Fig. 2). The correlation was maintained in patients with cirrhosis (r = 0.956; n = 6) but not in controls. No correlation was found between FHNC and fasting or average glucagon concentrations.

Before alanine infusion, the plasma amino-acid profile of cirrhotic patients was characterized by reduced levels of branched-chain amino acids and increased levels of aromatic amino acids (Table 3). After alanine infusion, nonsignificant changes in most plasma amino acids were observed, with the notable exceptions of branched-chain amino acids, aromatic amino acids and amino acids of the urea cycle, which decreased, and glutamine, which


y-Globulin Total bilirubin Prothrombin Esophageal Galactose elimination htipyrine clearance (gm.L-1) (mg . d-') activity (%) varices capacity (mg . kg-' . min-') (ml .min-')

21.8 14.1 13.4 23.0 15.6 24.8

18.8 2 4.6

11.8 I 1.8 9.9-13.2

1.2 1.5 1.5 1.0 3.0 1.3

1.6 t 0.6

0.7 f 0.2 0.4-1.1

92 61 61 66 51 55

64.3 f 13.3

94.0 5 12.0 70-100

Absent Large Medium Small Medium Large

4.78 4.06 2.66 3.81 4.45 2.22

3.66 f 0.92

6.47 f 0.64 5.91-7.29

23.4 12.9 12.1 26.0 19.2 11.9

17.6 t 5.6

37.9 t 4.6 33.4-44.9

TABLE 3. Plasma amino-acid concentrations at the beginning and at end of alanine infusion in the course of the paired experiments

Experiment Before alanine infusion After alanine infusion p Value

Spontaneous glucagon Alanine Control 347 f 86" 2,601 f 738 < 0.001

Cirrhotic 315 t 93 3,598 f 1,109 < 0.001 Glutamine Control 467 f 56 635 t 116 <0.01

Cirrhotic 369 * 107 503 f 148 <O.OOL BCAA Control 510 f 68 347 t 92 < 0.01

Cirrhotic 328 f 85' 318 t 79 NS AAA Control 141 t 15 102 5 22 < 0.01

Cirrhotic 189 * 35b 146 f 47 < 0.01 Ornithine Control 116 f 25 79 t 10 < 0.01

Cirrhotic 90 t 25 68 f 12 NS Arginine Control 124 t 30 92 f 34 < 0.01

Cirrhotic 83 2 18 68 t 24 NS

Alanine Control 247 f 101 2,247 2 458 < 0.001 Cirrhotic 296 f 70 2,825 t 730 < 0.0Ul

Glu tamine Control 222 t 57' 399 t 48' < 0.001 Cirrhotic 283 t 37 458 f 75 < 0.001

BCAA Control 441 t 119 361 f 122 < 0.01 Cirrhotic 364 2 68 329 c 50 NS

AAA Control 137 f 25 105 5 29 co.01 Cirrhotic 212 t 476 151 2 67 < 0.01

< 0.01 Ornithine Control 89 f 37 Cirrhotic 74 f 15 63 f 17 NS

Arginine Control 103 t 35 60 f 19 < 0.01 Cirrhotic 68 f 33 58 f 18 NS

Glucagon superinfusion

67 t 31

BCAA = sum of d i n e , isoleucine and leucine; AAA = sum of phenyldanine and tyrosine. "Data expressed as mean f S.D. (Fmol. L-l) . 'Significantly different from the corresponding control value. 'Significantly different from the corresponding value in the experiment carried out a t spontaneous glucagon concentrations.

increased. All these changes were more marked in control subjects. Glucagon Superinfusion. In the first 90 min, glu-

cagon concentrations increased to levels approximately four to six times higher than those found in the experiment carried out at spontaneous glucagon concentrations (Table 2). This immediately produced a re- markable decline in levels of a-amino nitrogen in both groups (controls, - 0.8 k 0.2 mmol- L- '; cirrhosis, - 0.5 k 0.3 mmol L and increased fasting glucose

(by 1.1 mmol.Lpl in controls but by only 0.8 mmol * L- ' in cirrhosis). At the beginning of alanine infusion, no differences in a-amino nitrogen, glucagon and glucose concentrations were observed between groups (Table 2). Insulin level was approximately doubled compared with values measured in the experiment without exogenous glucagon. During alanine infusion, blood glucose level decreased, insulin level did not change and glucagon level increased to similar end-of-infusion levels (approximately 10 times normal

32 FABBRIETAL HEPATOLOGY July 1993

Glucagan 0

Basal

CONTROLS

Basal Glucagan

CIRRHOSIS

FIG. 1. FHNC in control subjects (left, and in patients with cirrhosis fright) in the basal experiment at spontaneous glucagon concentrations and during glucagon superinfusion.

- , - C

0 w c3 I -*

1 4 I

51 I 10 20 30 40 50

Functional Hepatic Nitrogen Clearance (I/h)

FIG. 2. Relationship between FHNC and liver function (galactose elimination capacity and antipyrine clearance) in control subjects (0)

and patients with cirrhosis ( 0 ) in the experiment carried out at spontaneous glucagon concentrations. Correlation lines do not significantly differ when calculated separately in each group.

fasting values in controls and five times basal values in cirrhosis). Peak a-amino nitrogen concentrations were lower compared with those attained in the experiment without glucagon in control subjects but not in cirrhosis.

In controls, basal UNSR was significantly higher than that measured in the experiment without glucagon, but it was not different from the value measured in cirrhosis (52 5 23 mmol * hr- vs. 34 ? 22 mmol . hr-l) . In the

250

r Y 0 200 E E - 150

r z

fn 100 Z 3

50

0:

Contro I s Cirrhosis I

I 4.5 5.5 6.5 3.5 4.5 5.5 6.5

Alpha-amino Nitrogen (rnmol/l)

FIG. 3. Dynamics of a-amino nitrogen-to-urea nitrogen conversion in experiments carried out a t spontaneous glucagon concentrations (- - -) and during glucagon superinfusion (-1 in control subjects (left) and in patients with cirrhosis (right). Urea nitrogen synthesis rate increases with increasing a-amino nitrogen concentrations; the slope of the regression is the FHNC. Glucagon superinfusion produces a counterclockwise shift of the relationship (i.e., more urea is produced at any a-amino nitrogen concentration). The glucagon-induced change in dynamics is significantly greater in control subjects. The average equations of the regressions, where coefficients a and b are presented as mean (S.E.M.) of values obtained in individual experiments, are controls, basal: y = - 102.4 (16.1) + 35.3 (3.6). x; glucagon infusion: y = - 151.0 (22.4) + 57.7 (4.9) 'x ; cirrhosis, basal: y = -37.2 (11.0) + 23.2 (2.7).x; and glucagon infusion: y = 31.1 (16.3) + 26.7 (4.4) . X.

mmol hr - in controls (p vs. spontaneous glucagon, <0.05) and was lower by 30% in cirrhosis (140 -+ 30 mmol-hr-l ; p vs. control, ~ 0 . 0 2 ) . UNSR and the average a-amino nitrogen concentrations in each time period were linearly related (r = 0.83 to 0.951, but the average regression line was rotated counterclockwise compared with those from experiments carried out at spontaneous glucagon concentrations (Fig. 3). FHNC was 56% lower in cirrhosis (26.7 i- 10.7 L . hr- ' vs. 57.7 +- 11.0 L - hr-'; p < 0.01).

Compared with values obtained in the course of the experiment at spontaneous glucagon concentrations, FHNC was increased by glucagon infusion by 22.3 2 4.0 L . hr- ' (+63%) in controls and by only 3.5 ? 4.8 L * hr- ' ( + 15%) in cirrhosis (p vs. controls, < 0.01) (Fig. 1). In a single patient with near-normal liver function, FHNC increased by 11.4 L * hr - l.

FHNC measured at supraphysiological glucagon concentrations significantly correlated with galactose elimination and antipyrine clearance (r = 0.926 and 0.939, respectively; n = 11). The correlation with galactose elimination capacity was maintained in cirrhosis (r = 0.828; n = 61, whereas in controls FHNC correlated with antipyrine (r = 0.892; n = 5). Also, the increase in FHNC between paired experiments correlated with galactose elimination and antipyrine clearance (r = 0.905 and 0.964; Fig. 4) in the whole -

last period of alanine infusion, UNSR was 196 2 32 group of subjects.


Glucagon superinfusion significantly reduced plasma amino-acid concentrations, which, at the beginning of alanine infusion, were generally lower than those in the paired experiment without glucagon (Table 3). Re- markably, alanine and glutamine were reduced by 29% and 52% in controls and by 6% and 23% in cirrhosis. During alanine infusion, a further decline of most amino acids was observed (with the notable exception of glutamine). Again, the amino-acid-decreasing effect of alanine was more marked in control subjects, in whom levels of serine, threonine, proline, glycine, methionine, lysine and histidine significantly decreased also (not reported in detail).

DISCUSSION Our study shows that hepatic ureagenesis in response

to alanine infusion is significantly stimulated by glucagon infusion in control subjects, whereas in the presence of liver failure the stimulatory effect of glucagon is blunted or absent. Defective sensitivity to glucagon is related to decreased hepatocellular function, as proved by correlation with quantitative liver function tests.

In these experiments hepatic ureagenesis was studied by means of a procedure (calculation of hepatic nitrogen clearance) based on several assumptions that have been discussed extensively (6, 18). In the calculation of UNSR, intestinal hydrolysis of newly synthesized urea was considered a fixed fraction of total urea nitrogen excretion on the basis of the average values derived from the literature (20). Three patients were taking lactulose, which is likely to reduce gut urea hydrolysis (27). This may lead to overestimation of urea synthesis rate in cirrhosis, reducing the difference between patients and controls, but is not likely to be of any relevance in paired experiments.

Three factors play a major role in hepatic ureagenesis: substrate availability, hormonal drive and liver function (6).

Substrate availability was standardized by alanine infusion and by the calculation of the hepatic nitrogen clearance, as proposed by Vilstrup (6) and simplified by Bianchi et al. (18).

This study was intended to investigate the influence of hormonal drive, namely the glucagon drive, on hepatic nitrogen metabolism in patients with decreased hepatocellular function.

Glucagon plays an important role in amino-acid utilization and conversion into urea. Amino acids stimulate glucagon secretion (281, leading to hyperglucagonemia. High glucagon concentrations activate the enzymes of the urea cycle (2), synthesis of N-acetylglutamate (the activator of the feeder enzyme of car- bamoylphosphate synthetase) (29) and amino-acid transport systems across cell membranes (1). This results in increased hepatic utilization of amino acids for gluconeogenesis (30) and urea formation (31). In experimental animals (32) and in human beings (5) it has been shown that glucagon excess is associated with counter-

n

C n r = 0.905 E .- - 5 Y

2 E

3 r W

1 4 I

n 45 r = 0.964 C .-

E 25 -

0

U 54 I

Change in FHNC ( I /h) -5 0 5 10 15 20 25 30

FIG. 4. Relationship between glucagon-induced changes in FHNC and liver function (galactose elimination capacity and antipyrine clearance) in control subjects (0) and patients with cirrhosis (0).

Correlation lines do not differ significantly when calculated separately in each group.

clockwise shift of the relationship of UNSR to a-amino nitrogen (i.e., more urea is produced at any given amino-acid concentration). Such an effect was confirmed by data obtained in our healthy subjects (Fig. 3). The catabolic effects of glucagon on whole-body economy appear to be mediated by hepatic control of plasma amino-acid pools (33), which are expected to influence amino-acid release and uptake in the periphery.

In controls and in patients with cirrhosis, glucagon concentrations were made similar by exogenous glucagon administration. Glucagon infusion was calibrated to raise glucagon levels to a range approximately 10 times the normal concentration but only four to five times the average fasting concentration in cirrhotic patients, who occasionally have glucagon levels in this range. Such values raised FHNC by ~ 6 0 % in control subjects, in agreement with previous data obtained with a similar technique (51, but had scarce or no effect in patients with cirrhosis. Not only did glucagon fail to increase urea synthesis significantly, but it also had smaller effects on total a-amino nitrogen and individual plasma amino-acid concentrations.

Quantitative liver cell function is the last determinant of the capacity of the liver to convert a-amino nitrogen into urea nitrogen. Indeed, FHNC has been shown to depend on quantitative liver function (5, 7, 23, 271, and in these experiments we were able to confirm a correlation between FHNC and other quantitative liver function parameters. Most important, a direct correlation was shown between liver function - measured by galactose elimination capacity and antipyrine clearance-and the ability of glucagon to ameliorate the

34 FABBRIETAL. HEPATOLOGY July 1993

dynamics of a-amino nitrogen-to-urea nitrogen conversion. This suggests that, when the process is standardized for amino-acid and hormonal drive, liver function remains the major determinant of hepatic nitrogen metabolism and of the ability of the hepatic parenchyma to respond to the stimulatory effects of glucagon.

It might be argued that decreased responsiveness of the cirrhotic liver to glucagon infusion is due to previous submaximal stimulation by chronic hyperglucagonemia. However, in the high physiological range of glucagon concentrations observed in our experiments (five to 10 times basal values), Vilstrup et al. (5) reported a trend toward dose-dependence of functional nitrogen clearance on short-term glucagon stimulation. Were this the case, saturation would not be expected to occur in our patients with cirrhosis, in whom glucagon level was increased only two to three times, but a possible effect of chronic hyperglucagonemia cannot be ruled out.

Glucagon suppression, achieved with glucose infusion, can reduce alanine-stimulated hepatic nitrogen clearance (4); such down-regulation is less effective in cirrhosis (34). Unfortunately, glucagon secretion is not adequately suppressed by glucose in cirrhosis compared with that in healthy subjects (35). These two series of experiments clearly show that the short-term effects of glucagon on the hepatic parenchyma are lost in cirrhosis but do not explain the nature of the defect, which can only be addressed by studies with somato- statin and variable glucagon replacement. In any case, the decisive factor for decreased hepatic nitrogen clearance in cirrhosis is the extent of hepatocellular failure, which also prevents the response to exogenous glucagon .

Such glucagon unresponsiveness to nitrogen metabolism matches the failure of glucagon to increase glucose production (11, 12). In this study glucagon less effi- ciently increased glucose concentrations in cirrhosis in the first observation period compared with controls. The two defects may be basically the same (i.e., decreased amino-acid transport into the liver cell), with decreased substrate availability for both gluconeogenesis and urea synthesis.

A role of hyperglucagonemia has been put forward in the catabolic state and negative nitrogen balance of advanced cirrhosis (36). This hypothesis has been criticized (37) on the basis of the lack of glucagon to stimulate gluconeogenesis in cirrhosis. The finding that glucagon infusion also fails to produce effects on nitrogen metabolism, to stimulate urea synthesis or to induce nitrogen losses does not support a primary role of hyperglucagonemia in the muscle wasting and cachexia of cirrhosis.

We are indebted to Dr. Silvia Maselli and Dr. S. Stecanella, Farmacia, Policlinico S. Orsola, Bologna, Italy, for kindly preparing the alanine solution used in these experiments.

Acknowledgments:

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Documents

Unresponsiveness of hepatic nitrogen metabolism to glucagon infusion in patients with cirrhosis: Dependence on liver cell failure