Arginine is an important substrate in health and disease. It is a commonly held view that arginase-1 release from injured erythrocytes and hepatocytes leads to arginine breakdown; however, the true relationship between plasma arginase-1 concentration and activity has remained unaddressed. In the present study, blood was sampled from patients undergoing liver resection, a known cause of hepatocyte injury and arginase-1 release, to determine arginase-1, arginine and ornithine plasma levels. Arginase activity was assessed in vitro by measuring changes in arginine and ornithine plasma levels during incubation of plasma and whole-blood samples at 37 °C. Arginase-1 plasma levels increased 8–10-fold during liver resection, whereas arginine and ornithine levels remained unchanged. In accordance with these in vivo findings, arginine and ornithine levels remained unchanged in plasma incubated at 37 °C irrespective of the arginase-1 concentration. In contrast, arginine plasma levels in whole blood decreased significantly during incubation, with ornithine increasing stoichiometrically. These changes were irrespective of arginase-1 plasma levels and were explained by arginase activity present in intact erythrocytes. Next, plasma samples with 1000-fold normal arginase-1 concentrations were obtained from patients undergoing cadaveric liver transplantation. A significant decrease in arginine plasma levels occurred in vivo and in vitro. In contrast with commonly held views, moderately increased arginase-1 plasma levels do not affect plasma arginine. Very high plasma arginase-1 levels are required to induce potential clinically relevant effects.

INTRODUCTION

The amino acid arginine is an important substrate for protein synthesis and for the production of agmatine and creatine [1]. It is best known, however, as the precursor for the imunoregulatory and vasoactive molecule NO, although the conversion into NO represents only 1% of arginine plasma flux [2]. Quantitatively, arginine plasma flux is determined by arginine intake, endogenous arginine synthesis and protein breakdown on one side and protein synthesis and arginine catabolism by the enzyme arginase on the other side [3]. Arginase converts arginine into urea and ornithine, and arginase activity accounts for 10% of total plasma arginine turnover [4]. Arginase exists in two isoforms with only 58% sequence identity [5]. Arginase-1 is a cytosolic protein, predominantly found in the liver and to a lesser extent in erythrocytes [6]. Arginase-2 is located in mitochondria and more ubiquitously present [5], among others, in the kidneys and the spleen, but not in mature erythrocytes [6,7]. Hepatic arginase-1 activity serves urea synthesis and nitrogen homoeostasis. In the liver, ornithine is recycled to citrulline and back to arginine. Owing to compartmentalization of this urea cycle, plasma arginine is not a substrate for hepatic arginase-1 [8,9]. Ornithine generated outside the urea cycle can be converted into proline, an important constituent of collagen, and into polyamines, which are important for cell proliferation [1]. Changes in protein turnover, arginine intake or arginase activity can affect arginine plasma levels [3]. Arginine deficiency may result in microcirculatory disturbances, pulmonary and systemic hypertension [10,11], disturbed collagen synthesis and wound healing [12], impaired immune function [13,14] and in the onset of post-operative infections [15].

Injury to arginase-1-expressing cells, such as hepatocytes and erythrocytes, leads to arginase-1 release into the circulation and increased arginase-1 plasma levels [10,11,1619]. It is generally believed that such an increase leads to plasma arginine breakdown compromising arginine availability, potentially leading to microcirculatory dysfunction [10,11] and immune suppression [19,20]. Alternatively, regarding its effect on cell proliferation, arginine depletion by exogenous arginase may become a promising anticancer treatment [21]. The true relationship between the concentration of arginase-1 in plasma and its actual activity, however, has never been verified.

The aim of the present study was to investigate the effects of arginase-1 release on arginine plasma levels, using liver surgery (resection and transplantation) as a model of hepatocyte injury and arginase-1 release, and to establish the potential significance of circulating arginase-1 in arginine metabolism.

MATERIALS AND METHODS

Patients

Patients undergoing liver resection for secondary malignancies in an otherwise normal liver (n=16) were studied (Table 1). A routinely placed peripheral arterial catheter was used for blood sampling. Informed consent was obtained from every participant, and the research protocol was approved by the Institutional Medical Ethical Committee.

Table 1
Characteristics of patients undergoing liver resection

Values are medians (range). AST, aspartate aminotransferase; LDH, lactate dehydrogenase; IU, international unit.

In vivo studyIn vitro study
Surgical trauma Liver resection with intermittent Pringle manoeuvre Liver mobilization/manipulation prior to liver resection 
n 10 
Gender (n) (male/female) 5/5 3/3 
Age (years) 50 (33–75) 54 (36–74) 
Colorectal liver metastases/other 8/2 6/0 
liver metastases (n  
AST (IU/l) 23 (7–37) 34 (8–59) 
LDH (IU/l) 360 (295–490) 387 (351–422) 
Creatinine (μmol/l) 103 (67–118) 93 (56–106) 
In vivo studyIn vitro study
Surgical trauma Liver resection with intermittent Pringle manoeuvre Liver mobilization/manipulation prior to liver resection 
n 10 
Gender (n) (male/female) 5/5 3/3 
Age (years) 50 (33–75) 54 (36–74) 
Colorectal liver metastases/other 8/2 6/0 
liver metastases (n  
AST (IU/l) 23 (7–37) 34 (8–59) 
LDH (IU/l) 360 (295–490) 387 (351–422) 
Creatinine (μmol/l) 103 (67–118) 93 (56–106) 

Arginase-1 protein release and indices of in vivo arginase enzyme activity during liver resection

Arginase-1 release and its effect on arginine and ornithine levels were studied in vivo in ten patients undergoing hepatectomy with intermittent hepatic inflow occlusion (Pringle manoeuvre) [22]. Arterial blood was sampled pre-operatively, before liver transection, before and after each event of an intermittent Pringle manoeuvre (2×15 min of ischemia and 5 min of reperfusion) and 90 min post-operatively. Blood was collected in pre-chilled heparinized vacuum tubes (Becton Dickinson), immediately placed on ice and processed as described below.

In vitro arginase enzyme activity in whole blood and plasma after liver manipulation

To study the effect of plasma arginase activity, separate from other processes regulating arginine levels in vivo, plasma samples with various arginase-1 levels were incubated. In addition, arginase activity was assessed in corresponding whole-blood samples. Arterial blood was obtained from six patients undergoing liver resection, pre-operatively and after liver manipulation (before liver transection), when, based upon prior experience, peak arginase-1 concentrations were already expected [23].

Pre-incubation blood processing

Blood was collected in two 4 ml heparin tubes (Becton Dickinson). One tube was centrifuged (4000 g, 20 °C, 5 min) to obtain plasma. The plasma was removed and transferred to clean tubes for incubation. The whole-blood samples were incubated without any further processing.

Incubation

Incubations were performed immediately after sampling. Plasma and whole-blood samples were divided into three aliquots per fraction which were incubated for 0, 20 or 40 min at 37 °C. Thereafter, samples were placed on ice and processed immediately as described below. Arginase-1, arginine, ornithine and other amino acid concentrations were measured as described below. Arginase activity was expressed as the increment in ornithine concentration/min. Since kinetic assays like these do not discriminate between the activities of different isoenzymes, the term arginase activity will be used without further specification when referring to the data from this assay.

Arginase-1 protein release and plasma arginase enzyme activity after liver transplantation

Roth et al. [16] described a decrease in plasma arginine levels from ±100 μmol/l to ±4 μmol/l within 30 min following cadaveric liver transplantation, which was ascribed to arginase release from the graft and overwhelming plasma arginase activity. Ethical permission was granted to include four patients undergoing cadaveric liver transplantation at the University Hospital, Leuven, Belgium (Table 2) as optimal positive controls for the present study. Arterial blood was drawn into heparin tubes (Becton Dickinson) pre-operatively, at the end of the anhepatic phase, and 3, 20 and 60 min after reperfusion of the graft. Whole blood was centrifuged at 4000 g for 5 min. Argininase-1, arginine, ornithine and other amino acid concentrations were measured in all samples. Plasma obtained 3 min after reperfusion was incubated at 37 °C for 25–40 (mean 30) min to assess plasma arginase activity as described above.

Table 2
Characteristics of the patients undergoing liver transplantation

AST, aspartate aminotransferase; HCC, hepatocellular carcinoma; IU, international unit; LDH, lactate dehydrogenase; Pre, pre-operative; 3 min rep, 3 min after reperfusion.

AST (IU/l)LDH (IU/l)Creatinine (μmol/l)
PatientGenderAge (years)Indication for transplantationCold ischaemia time (h:min)Pre3 min repPre3 min repPre3 min rep
Female 23 Acute Budd–Chiari 10:51 1956 1786 2417 4680 410 318 
Male 55 Alcoholic cirrhosis+HCC 8:57 40 479 274 1544 62 61 
Male 61 Alcoholic cirrhosis 11:26 71 458 572 1582 62 68 
Male 73 Hepatitis C cirrhosis+HCC 12:29 68 290 329 855 21 98 
AST (IU/l)LDH (IU/l)Creatinine (μmol/l)
PatientGenderAge (years)Indication for transplantationCold ischaemia time (h:min)Pre3 min repPre3 min repPre3 min rep
Female 23 Acute Budd–Chiari 10:51 1956 1786 2417 4680 410 318 
Male 55 Alcoholic cirrhosis+HCC 8:57 40 479 274 1544 62 61 
Male 61 Alcoholic cirrhosis 11:26 71 458 572 1582 62 68 
Male 73 Hepatitis C cirrhosis+HCC 12:29 68 290 329 855 21 98 

Relationship between arginase activity and arginase-1 concentration in plasma

Plasma arginase activity at physiological arginine concentrations

Aliquots of a plasma sample from one liver transplant recipient, obtained immediately following reperfusion (arginase-1 concentration, 25 μg/ml), were mixed with aliquots of a plasma sample obtained from a healthy subject (arginase-1 concentration below detection limit of 10 ng/ml). By this means, an arginase-1 dilution curve ranging from 25 μg/ml to 195 ng/ml was created. Before mixing, PBS (pH 7.4) containing 85 mmol/l arginine was added to the ‘healthy’ plasma aliquots. The volume of arginine-enriched PBS added to each aliquot was adjusted so that the final arginine concentration after mixing equalled 85 μmol/l in each sample. Accordingly, plasma samples were created containing various arginase-1 and equal arginine concentrations, the maximum concentration of PBS in these samples was <0.1%. All samples were immediately incubated at 37 °C for 0, 5 or 30 min.

Plasma arginase activity at above-Km arginine concentrations

In a similar fashion as described above, plasma aliquots containing various concentrations of arginase-1 and 20 mmol/l arginine were created. However, in this case, arginine was dissolved directly in the ‘healthy’ plasma aliquots to avoid dilution of the plasma with PBS. Eventually plasma samples were created containing 15000, 1500 or 150 ng/ml arginase-1 with 20 mmol/l arginine. These samples were immediately incubated at 37 °C for 0, 5 or 30 min.

Arginase-1 concentration in erythrocytes

Blood was obtained from five healthy volunteers in vacuum tubes containing EDTA (Becton Dickinson). To remove plasma and mononuclear cells, a protocol described previously was applied using a separation fluid (Lymphoprep 1.077; Axis-Shield) [24]. A total of 1 ml of packed erythrocytes was added to 74 ml of distilled water and gently shaken for 10 min to induce 100% haemolysis [25]. The homogenate was centrifuged (4000 g, 20 °C, 15 min), and the supernatant was assayed for arginase-1.

Post-incubation sample processing and laboratory analysis

Whole-blood samples were centrifuged (4000 g, 4 °C, 5 min) to obtain plasma. A total of 100 μl of plasma was deproteinized with 8 mg of SSA (sulfosalicylic acid) and stored at −80 °C, and the remainder was stored untreated (−80 °C). Amino acid levels were analysed in SSA-deproteinized samples by HPLC, as described previously [26]. Arginase-1 concentrations were measured in untreated samples using an ELISA [27] (kindly provided by Hycult Biotechnology). The detection limit of this assay is 10 ng/ml arginase-1.

Statistics

Changes in amino acid and arginase-1 concentrations in vivo and in vitro were tested using a paired Student's t test or one-way ANOVA for repeated measures when more than two serial observations were available. Statistical calculations were made using Prism 4.0 for Windows (GraphPad Software). Results are expressed as means (S.E.M.), with patient characteristics expressed as medians (range). A P value <0.05 was considered to indicate statistical significance.

RESULTS

Effects of liver manipulation and warm ischemia

As expected, mean arginase-1 plasma levels increased significantly during liver manipulation (8-fold), before hepatic inflow occlusion (Figure 1A). Despite this, plasma concentrations of arginine and ornithine remained unchanged during the same period (Figure 1B). During inflow occlusion, no further significant changes in arginase-1 plasma levels were observed (Figure 1A). At the same time, plasma levels of arginine (and most other amino acids; results not shown) increased. Ornithine concentrations remained unchanged (Figure 1B). At 90 min post-operatively, the mean arginase-1 plasma concentration decreased to 17% of the last measured intra-operative value. From this, an arginase-1 plasma half-life of less than 1 h was calculated.

Arterial arginase-1 (A) and arginine and ornithine (B) concentrations

Figure 1
Arterial arginase-1 (A) and arginine and ornithine (B) concentrations

Arterial arginase-1 (A) and arginine and ornithine (B) concentrations were measured in ten patients undergoing partial hepatectomy with an intermittent Pringle manoeuvre [15 min of clamping and 5 min of reperfusion; clamping (C) and unclamping (U) are indicated by arrows]. Arginase-1 levels increased from the start of the procedure (P=0.0005), without affecting arginine levels (P=0.43). A significant increase in arginine levels was observed during hepatic pedicle clamping (P<0.0001), most probably due to abolishment of hepatic amino acid clearance. Values are means (S.E.M.).

Figure 1
Arterial arginase-1 (A) and arginine and ornithine (B) concentrations

Arterial arginase-1 (A) and arginine and ornithine (B) concentrations were measured in ten patients undergoing partial hepatectomy with an intermittent Pringle manoeuvre [15 min of clamping and 5 min of reperfusion; clamping (C) and unclamping (U) are indicated by arrows]. Arginase-1 levels increased from the start of the procedure (P=0.0005), without affecting arginine levels (P=0.43). A significant increase in arginine levels was observed during hepatic pedicle clamping (P<0.0001), most probably due to abolishment of hepatic amino acid clearance. Values are means (S.E.M.).

In vitro arginase activity

Blood was sampled from six patients undergoing liver resection pre-operatively and after liver manipulation. Pre-operative arterial arginase-1 plasma levels were 18.1 (8.0) ng/ml. In line with the results mentioned above, arginase-1 plasma levels increased significantly during liver manipulation [10-fold to 184 (54) ng/ml], without affecting arginine and ornithine levels in vivo (P=0.72 and P=0.16 respectively; Figure 2).

In vitro arginase activity in plasma (A and B) and whole blood (C and D)

Figure 2
In vitro arginase activity in plasma (A and B) and whole blood (C and D)

Blood was drawn from six patients undergoing liver resection immediately before surgery (A and C) and after liver manipulation (B and D). The mean (S.E.M.) pre-operative arginase-1 plasma concentration was 18.2 (8.0) ng/ml, which increased to 184 (54) ng/ml following liver manipulation (10-fold increase; P=0.028), without affecting arginine and ornithine levels in vivo (P=0.72 and P=0.16 respectively). Plasma, whole blood and erythrocytes suspended in PBS+80 μmol/l arginine obtained before and during surgery were incubated for 0, 20 and 40 min at 37 °C. No significant changes in arginine and ornithine concentrations were found in plasma (A and B) irrespective of the amount of arginase-1 in the samples. Arginine concentrations in whole blood (C and D) decreased significantly. Decreasing arginine levels were accompanied by stoichiometric increases in ornithine levels in all cases, indicating arginase activity. The amount of extracellular arginase-1 in the incubated whole-blood samples did not influence the amount of ornithine formed. *P<0.005. ns, not significant.

Figure 2
In vitro arginase activity in plasma (A and B) and whole blood (C and D)

Blood was drawn from six patients undergoing liver resection immediately before surgery (A and C) and after liver manipulation (B and D). The mean (S.E.M.) pre-operative arginase-1 plasma concentration was 18.2 (8.0) ng/ml, which increased to 184 (54) ng/ml following liver manipulation (10-fold increase; P=0.028), without affecting arginine and ornithine levels in vivo (P=0.72 and P=0.16 respectively). Plasma, whole blood and erythrocytes suspended in PBS+80 μmol/l arginine obtained before and during surgery were incubated for 0, 20 and 40 min at 37 °C. No significant changes in arginine and ornithine concentrations were found in plasma (A and B) irrespective of the amount of arginase-1 in the samples. Arginine concentrations in whole blood (C and D) decreased significantly. Decreasing arginine levels were accompanied by stoichiometric increases in ornithine levels in all cases, indicating arginase activity. The amount of extracellular arginase-1 in the incubated whole-blood samples did not influence the amount of ornithine formed. *P<0.005. ns, not significant.

Plasma

Incubation of pre-operatively collected plasma samples for 40 min at 37 °C did not lead to changes in arginine and ornithine concentrations (P=0.26; Figure 2A). In accordance with the in vivo findings, no significant changes in arginine and ornithine concentrations were observed during the incubation of plasma samples containing a 10-fold increased arginase-1 concentration (P=0.33; Figure 2B). Plasma concentrations of other amino acids were not affected by in vitro incubation (Table 3).

Table 3
Changes in plasma amino acid concentrations during incubation of plasma

Results are presented as means (S.E.M.), and are pooled data from all plasma incubation experiments. Plasma was obtained from patients undergoing liver resection before liver manipulation (n=6), after liver manipulation (n=6) and after liver transplantation (n=4). Arterial concentrations before incubation are given as well as absolute (μmol/l) and relative (%) increases at the end of the incubation (mean, 37.5 min). Results were similar for all three experiments, except for changes in arginine and ornithine concentrations in plasma obtained after liver transplantation. Changes in arginine and ornithine concentrations during the various experiments are shown in Figures 2 and 3(C). The sum of arginine and ornithine concentrations remained unchanged during all experiments. Absolute and relative changes were tested compared with time zero by a one-sample Student's t test. ΣAA, sum of all amino acids; Σarginine+ornithine, sum of arginine and ornithine concentrations.

End of incubation
Amino acidBefore incubation (μmol/l)Absolute increase (μmol/l)P valueRelative increase (%)P value
Glutamate 114 (9.3) 2.3 (2.9) 0.451 3.0 (3.1) 0.350 
Asparagine 68 (11.7) 4.3 (2.2) 0.083 4.6 (2.8) 0.130 
Serine 137 (14.3) 2.6 (2.7) 0.355 1.3 (1.8) 0.481 
Glutamine 626 (52.9) 11.6 (12.5) 0.372 1.7 (2.3) 0.481 
Histidine 188 (105.1) 0.6 (5.3) 0.915 −5.7 (9.8) 0.573 
Glycine 329 (40.3) 9.8 (8.7) 0.287 8.8 (12.5) 0.496 
Threonine 155 (18.9) 0.5 (4.4) 0.911 −1.3 (2.8) 0.653 
Citrulline 40 (2.7) 0.7 (1.0) 0.526 1.3 (2.3) 0.582 
Alanine 404 (38.5) 12.8 (8.3) 0.148 3.2 (2.2) 0.187 
Taurine 61 (7.5) 2.1 (1.2) 0.113 10.2 (8.2) 0.237 
α-Amino butyric acid 17 (1.3) 0.7 (0.4) 0.136 3.7 (2.3) 0.140 
Tyrosine 84 (15.6) 6.6 (5.4) 0.246 5.1 (4.4) 0.272 
Valine 156 (12.0) 7.5 (5.1) 0.173 3.2 (2.7) 0.273 
Methionine 38 (11.1) 1.5 (0.8) 0.098 2.8 (2.3) 0.265 
Isoleucine 52 (3.5) 2.3 (1.5) 0.157 3.5 (2.6) 0.207 
Phenylalanine 68 (11.5 2.0 (1.2) 0.122 2.2 (1.7) 0.213 
Tryptophan 51 (7.5) 1.3 (1.3) 0.370 3.7 (3.1) 0.251 
Leucine 101 (8.5) 4.3 (2.2) 0.075 3.2 (2.0) 0.134 
Lysine 212 (19.0) 8.0 (5.8) 0.193 2.8 (2.6) 0.313 
ΣAA 3073 (291.4) 90.3 (61.7) 0.171 2.3 (2.0) 0.283 
ΣArginine+ornithine 161 (10.0) 4.8 (3.7) 0.219 1.7 (2.1) 0.424 
End of incubation
Amino acidBefore incubation (μmol/l)Absolute increase (μmol/l)P valueRelative increase (%)P value
Glutamate 114 (9.3) 2.3 (2.9) 0.451 3.0 (3.1) 0.350 
Asparagine 68 (11.7) 4.3 (2.2) 0.083 4.6 (2.8) 0.130 
Serine 137 (14.3) 2.6 (2.7) 0.355 1.3 (1.8) 0.481 
Glutamine 626 (52.9) 11.6 (12.5) 0.372 1.7 (2.3) 0.481 
Histidine 188 (105.1) 0.6 (5.3) 0.915 −5.7 (9.8) 0.573 
Glycine 329 (40.3) 9.8 (8.7) 0.287 8.8 (12.5) 0.496 
Threonine 155 (18.9) 0.5 (4.4) 0.911 −1.3 (2.8) 0.653 
Citrulline 40 (2.7) 0.7 (1.0) 0.526 1.3 (2.3) 0.582 
Alanine 404 (38.5) 12.8 (8.3) 0.148 3.2 (2.2) 0.187 
Taurine 61 (7.5) 2.1 (1.2) 0.113 10.2 (8.2) 0.237 
α-Amino butyric acid 17 (1.3) 0.7 (0.4) 0.136 3.7 (2.3) 0.140 
Tyrosine 84 (15.6) 6.6 (5.4) 0.246 5.1 (4.4) 0.272 
Valine 156 (12.0) 7.5 (5.1) 0.173 3.2 (2.7) 0.273 
Methionine 38 (11.1) 1.5 (0.8) 0.098 2.8 (2.3) 0.265 
Isoleucine 52 (3.5) 2.3 (1.5) 0.157 3.5 (2.6) 0.207 
Phenylalanine 68 (11.5 2.0 (1.2) 0.122 2.2 (1.7) 0.213 
Tryptophan 51 (7.5) 1.3 (1.3) 0.370 3.7 (3.1) 0.251 
Leucine 101 (8.5) 4.3 (2.2) 0.075 3.2 (2.0) 0.134 
Lysine 212 (19.0) 8.0 (5.8) 0.193 2.8 (2.6) 0.313 
ΣAA 3073 (291.4) 90.3 (61.7) 0.171 2.3 (2.0) 0.283 
ΣArginine+ornithine 161 (10.0) 4.8 (3.7) 0.219 1.7 (2.1) 0.424 

Whole blood

Arginase-1 plasma levels in incubated whole blood remained stable (results not shown), ruling out a potential increase in arginase activity due to haemolysis. After 40 min of incubation, a significant decrease in arginine concentrations with a concomitant increase in ornithine concentrations was found in plasma of all the whole-blood samples, irrespective of the amount of arginase-1 in the plasma (Figure 2C and 2D). These highly significant changes were not found for other amino acids (Table 4).

Table 4
Changes in plasma amino acid concentrations during incubation of whole blood

Results are presented as means (S.E.M), and are pooled data for all whole-blood incubation experiments. Whole blood was obtained from patients undergoing liver resection before (n=6) and after (n=6) liver manipulation. Arterial concentrations before incubation are given as well as absolute (μmol/l) and relative (%) increases at the end of the incubation (mean, 37.5 min). Results were similar for both experiments. Absolute and relative changes were compared with time zero by a one-sample Student's t test. ΣAA, sum of all amino acids; Σarginine+ornithine, sum of arginine and ornithine concentrations.

End of incubation
Amino acidBefore incubation (μmol/l)Absolute increase (μmol/l)P valueRelative increase (%)P value
Glutamate 109 (6.8) 9.6 (4.7) 0.078 8.3 (4.0) 0.077 
Asparagine 43 (2.0) 3.8 (1.7) 0.066 7.7 (3.8) 0.079 
Serine 118 (10.7) 3.6 (5.3) 0.514 1.7 (4.4) 0.711 
Glutamine 527 (9.3) 3.1 (23.6) 0.898 0.5 (4.5) 0.924 
Histidine 61 (13.7) 6.3 (4.4) 0.196 6.6 (5.2) 0.247 
Glycine 305 (53.0) 45.9 (41.8) 0.308 55.4 (41.3) 0.221 
Threonine 117 (9.5) 4.1 (5.0) 0.433 2.0 (4.8) 0.692 
Citrulline 39 (3.0) 1.0 (1.1) 0.394 2.2 (2.7) 0.437 
Arginine 68 (3.1) −18.3 (2.2) <0.001 −26.7 (3.1) <0.001 
Alanine 321 (26.0) 28.4 (14.6) 0.094 9.3 (4.2) 0.062 
Taurine 58 (5.3) −1.4 (10.9) 0.903 3.2 (16.3) 0.850 
α-Amino butyric acid 14 (0.8) 1.3 (0.5) 0.049 8.4 (3.4) 0.043 
Tyrosine 49 (2.9) 2.1 (1.7) 0.239 3.5 (3.3) 0.327 
Valine 148 (11.0) 9.3 (5.3) 0.127 5.0 (3.2) 0.161 
Methionine 16 (0.9) 0.5 (0.5) 0.351 2.3 (3.0) 0.473 
Isoleucine 46 (3.6) 2.9 (1.6) 0.111 4.6 (3.3) 0.209 
Phenylalanine 43 (2.6) 1.9 (1.3) 0.201 4.0 (3.0) 0.214 
Tryptophan 34 (4.0) 2.1 (2.3) 0.391 7.8 (8.7) 0.401 
Leucine 85 (7.4) 7.3 (2.9) 0.042 7.1 (2.9) 0.045 
Ornithine 73 (5.9) 37.6 (7.5) <0.001 45.1 (5.8) <0.001 
Lysine 167 (14.1) 19.0 (11.2) 0.132 10.0 (5.2) 0.094 
ΣAA 2445 (111.6) 172.1 (105.1) 0.145 6.9 (4.3) 0.153 
ΣArginine+ornithine 145 (8.5) 19.3 (7.4) 0.024 12.0 (4.1) 0.015 
End of incubation
Amino acidBefore incubation (μmol/l)Absolute increase (μmol/l)P valueRelative increase (%)P value
Glutamate 109 (6.8) 9.6 (4.7) 0.078 8.3 (4.0) 0.077 
Asparagine 43 (2.0) 3.8 (1.7) 0.066 7.7 (3.8) 0.079 
Serine 118 (10.7) 3.6 (5.3) 0.514 1.7 (4.4) 0.711 
Glutamine 527 (9.3) 3.1 (23.6) 0.898 0.5 (4.5) 0.924 
Histidine 61 (13.7) 6.3 (4.4) 0.196 6.6 (5.2) 0.247 
Glycine 305 (53.0) 45.9 (41.8) 0.308 55.4 (41.3) 0.221 
Threonine 117 (9.5) 4.1 (5.0) 0.433 2.0 (4.8) 0.692 
Citrulline 39 (3.0) 1.0 (1.1) 0.394 2.2 (2.7) 0.437 
Arginine 68 (3.1) −18.3 (2.2) <0.001 −26.7 (3.1) <0.001 
Alanine 321 (26.0) 28.4 (14.6) 0.094 9.3 (4.2) 0.062 
Taurine 58 (5.3) −1.4 (10.9) 0.903 3.2 (16.3) 0.850 
α-Amino butyric acid 14 (0.8) 1.3 (0.5) 0.049 8.4 (3.4) 0.043 
Tyrosine 49 (2.9) 2.1 (1.7) 0.239 3.5 (3.3) 0.327 
Valine 148 (11.0) 9.3 (5.3) 0.127 5.0 (3.2) 0.161 
Methionine 16 (0.9) 0.5 (0.5) 0.351 2.3 (3.0) 0.473 
Isoleucine 46 (3.6) 2.9 (1.6) 0.111 4.6 (3.3) 0.209 
Phenylalanine 43 (2.6) 1.9 (1.3) 0.201 4.0 (3.0) 0.214 
Tryptophan 34 (4.0) 2.1 (2.3) 0.391 7.8 (8.7) 0.401 
Leucine 85 (7.4) 7.3 (2.9) 0.042 7.1 (2.9) 0.045 
Ornithine 73 (5.9) 37.6 (7.5) <0.001 45.1 (5.8) <0.001 
Lysine 167 (14.1) 19.0 (11.2) 0.132 10.0 (5.2) 0.094 
ΣAA 2445 (111.6) 172.1 (105.1) 0.145 6.9 (4.3) 0.153 
ΣArginine+ornithine 145 (8.5) 19.3 (7.4) 0.024 12.0 (4.1) 0.015 

Plasma arginase activity after liver transplantation

In vivo arginase-1 levels and indices of arginase activity

After reperfusion, plasma arginase-1 levels increased rapidly to 1000-fold normal values (Figure 3A). In addition, we observed a rapid decline in plasma arginine levels with a concomitant increase in the plasma levels of ornithine (Figure 3A). A one-phase exponential decay curve fitted along the mean arginase-1 concentrations 3, 20 and 60 min post-reperfusion resulted in a calculated plasma half life of approx. 40 min. Within 3 min following reperfusion, in vivo arginine plasma levels were relatively stabilized (Figure 3B). Plasma concentrations of other amino acids that depend on the liver for their plasma clearance, such as alanine and methionine, also declined following reperfusion of the liver, although not as steeply as arginine concentrations (Figure 3B).

Plasma arginase activity after liver transplantation
Figure 3
Plasma arginase activity after liver transplantation

(A) Arterial arginase-1, arginine (arg) and ornithine (orn) concentrations were measured in four patients undergoing cadaveric liver transplantation. Pre-operative (preop) arginase-1 levels were elevated above reference values, reflecting underlying liver disease eventually necessitating liver transplantation. Immediately following reperfusion, a large increase in plasma arginase-1 levels occurred (P=0.008). This was accompanied by a rapid decrease in arginine levels (P<0.0001) and a concomitant increase in ornithine levels (P=0.0003). (B) The decline in plasma arginine levels was most marked in the first 3 min, whereafter arginine levels remained relatively stable. (C) Incubation of plasma samples drawn 3 min after reperfusion of the liver graft [containing a mean (S.E.M.) arginase-1 concentration of 13 (4.2) μg/ml] led to a significant decrease in arginine levels with a concomitant increase in ornithine levels (P=0.002), proving plasma arginase activity.

Figure 3
Plasma arginase activity after liver transplantation

(A) Arterial arginase-1, arginine (arg) and ornithine (orn) concentrations were measured in four patients undergoing cadaveric liver transplantation. Pre-operative (preop) arginase-1 levels were elevated above reference values, reflecting underlying liver disease eventually necessitating liver transplantation. Immediately following reperfusion, a large increase in plasma arginase-1 levels occurred (P=0.008). This was accompanied by a rapid decrease in arginine levels (P<0.0001) and a concomitant increase in ornithine levels (P=0.0003). (B) The decline in plasma arginine levels was most marked in the first 3 min, whereafter arginine levels remained relatively stable. (C) Incubation of plasma samples drawn 3 min after reperfusion of the liver graft [containing a mean (S.E.M.) arginase-1 concentration of 13 (4.2) μg/ml] led to a significant decrease in arginine levels with a concomitant increase in ornithine levels (P=0.002), proving plasma arginase activity.

In vitro arginase activity after liver transplantation

Incubation of plasma sampled 3 min after liver transplantation [arginase-1, 13.2 (4.2 μg/ml)] at 37 °C for 30 min resulted in a significant decrease in plasma arginine concentration [from 61.1 (7.4) to 28.4 (6.7) μmol/l]. This was accompanied by a similar increase in ornithine concentration [from 125.2 (6.6) to 163.7 (10.4) μmol/l; P=0.002; Figure 3C]. Other amino acids remained unaffected (Table 3).

Relationship between arginase enzyme activity and arginase-1 protein concentrations in plasma

Plasma arginase activity at physiological arginine concentrations

Plasma samples containing different arginase-1 levels were incubated for 5 and 30 min at 37 °C in the presence of 85 μmol/l arginine. At this arginine concentration, the relationship between arginase-1 concentration and arginase activity appeared to be non-linear, with a lower specific activity (activity/unit of enzyme) at higher arginase-1 concentrations. Below an arginase-1 concentration of 1.6 μg/ml, however, there was in fact a linear relationship between arginase-1 concentration and arginase activity (r2=0.99) (Figure 4A). After 30 min of incubation with an initial arginine concentration of 85 μmol/l, arginine plasma levels decreased dependent on the arginase-1 level. At arginase-1 levels below 2 μg/ml and an initial arginine concentration of 85 μmol/l, arginine levels decreased less than 10% within 30 min (Figure 4B).

Relationship between arginase enzyme activity and arginase-1 protein concentrations in plasma
Figure 4
Relationship between arginase enzyme activity and arginase-1 protein concentrations in plasma

Plasma samples with various arginase-1 concentrations were created by mixing plasma from a liver transplant recipient (arginase-1 concentration, 25 μg/ml) and from a healthy volunteer (arginase-1 concentration, below 10 ng/ml). Starting arginine levels were adjusted by adding arginine, and samples were incubated at 37 °C. (A) Plasma samples were incubated for 5 min in the presence of 85 μmol/l arginine, which is approximately the normal arginine concentration in human plasma. The results show that arginase activity was related to arginase-1 concentration; however, the formation of ornithine at the highest arginase-1 concentrations was rapidly limited by decreasing substrate availability. The broken line indicates the theoretical relationship between arginase-1 concentration and arginase activity at a constant arginine concentration of 85 μmol/l. (B) Plasma samples with various concentrations of arginase-1 were incubated in the presence of 85 μmol/l arginine for 20 min. During this period arginine plasma levels decrease was dependent on the arginase-1 plasma level. At arginase-1 levels below 2000 ng/ml, the decrease in arginine level in 20 min was below 10%. Below an arginine plasma level of 50 μmol/l, arginase activity rapidly becomes limited by substrate availability. Lx, mean arginase-1 levels reached during liver resection; LTx, mean arginase-1 levels reached after liver transplantation. (C) Plasma samples with varying concentrations of arginase-1 were incubated in the presence of 20 mmol/l arginine, which maintained substrate availability above the Km values during the incubation. In this experiment, the ornithine formation rate remained unchanged, showing that the intrinsic activity of the enzyme was maintained during the incubations.

Figure 4
Relationship between arginase enzyme activity and arginase-1 protein concentrations in plasma

Plasma samples with various arginase-1 concentrations were created by mixing plasma from a liver transplant recipient (arginase-1 concentration, 25 μg/ml) and from a healthy volunteer (arginase-1 concentration, below 10 ng/ml). Starting arginine levels were adjusted by adding arginine, and samples were incubated at 37 °C. (A) Plasma samples were incubated for 5 min in the presence of 85 μmol/l arginine, which is approximately the normal arginine concentration in human plasma. The results show that arginase activity was related to arginase-1 concentration; however, the formation of ornithine at the highest arginase-1 concentrations was rapidly limited by decreasing substrate availability. The broken line indicates the theoretical relationship between arginase-1 concentration and arginase activity at a constant arginine concentration of 85 μmol/l. (B) Plasma samples with various concentrations of arginase-1 were incubated in the presence of 85 μmol/l arginine for 20 min. During this period arginine plasma levels decrease was dependent on the arginase-1 plasma level. At arginase-1 levels below 2000 ng/ml, the decrease in arginine level in 20 min was below 10%. Below an arginine plasma level of 50 μmol/l, arginase activity rapidly becomes limited by substrate availability. Lx, mean arginase-1 levels reached during liver resection; LTx, mean arginase-1 levels reached after liver transplantation. (C) Plasma samples with varying concentrations of arginase-1 were incubated in the presence of 20 mmol/l arginine, which maintained substrate availability above the Km values during the incubation. In this experiment, the ornithine formation rate remained unchanged, showing that the intrinsic activity of the enzyme was maintained during the incubations.

Plasma arginase activity at above-Km arginine concentrations

Plasma with various amounts of arginase-1 was incubated in the presence of 20 mmol/l arginine, which is well above the Km for arginase-1. This resulted in an enduring linear ornithine formation (Figure 4C), showing that the intrinsic activity of the enzyme in isolated plasma was preserved during in vitro incubation.

Arginase-1 concentration in erythrocytes

Erythrocyte arginase-1 concentration, measured by ELISA, was 17.0 (1.1) μg/ml of erythrocytes.

DISCUSSION

Increased plasma arginase-1 activity is frequently suggested as a cause of low arginine plasma levels in patients with hepatocellular or erythrocyte injury [10,11,16,17,20,28,29]. This could be clinically relevant, since low arginine levels may induce immunological and microcirculatory dysfunction [14,30,31]. The results from the present study, however, show that a potentially relevant effect of plasma arginase-1 on arginine metabolism only occurs when very high arginase-1 concentrations are reached. We were able to study the relationship between plasma arginase-1 levels and plasma arginase activity by the application of a recently developed ELISA [27] that allows exact quantification of the arginase-1 concentration in plasma.

Arginase-1 was released from the liver into the plasma in patients undergoing liver resection, leading to an 8-fold increase in circulating arginase-1, without affecting arginine and ornithine plasma levels. These findings show no evidence for increased arginase activity, despite increased arginase-1 plasma levels. From these results alone, however, it cannot be ruled out that actual arginase activity was compensated for by other mechanisms regulating plasma arginine and ornithine levels. Plasma arginine levels are regulated in vivo by protein breakdown and endogenous arginine synthesis. The latter occurs in the kidney, where citrulline, derived from intestinal glutamine metabolism, becomes converted into arginine [9,32]. We have demonstrated recently that endogenous arginine synthesis remains unchanged during liver surgery [32]. Quantitatively, arginine clearance is determined primarily by protein synthesis. This is illustrated by the increased plasma levels of arginine and most other amino acids during hepatic inflow occlusion, reflecting abolishment of hepatic protein synthesis. Ornithine concentrations remained unchanged, which is in line with the absence of physiological hepatic ornithine uptake [9].

To study plasma arginase activity isolated from in vivo regulatory mechanisms, plasma and whole-blood samples were incubated in vitro. In agreement with the in vivo results, no significant changes in arginine and ornithine concentrations were found during incubation of plasma samples with a mean arginase-1 concentration of 185 ng/ml. To explore whether plasma arginase activity was dependent on factors present in whole blood, but not in plasma, whole-blood samples were incubated at 37 °C for 40 min. A significant increase in ornithine was found, accompanied by a similar decrease in arginine. No changes were found in the plasma concentrations of other amino acids such as glutamate, an ornithine precursor, or citrulline, an arginine product. This strongly suggests that the observed changes actually specifically reflect arginase activity. Whole-blood arginase activity, however, was not related to the arginase-1 plasma concentration, which underlines that plasma arginase-1 does not affect plasma arginine levels. More likely, whole-blood arginase activity occurs in the cellular fraction (erythrocytes and/or leucocytes).

Several studies have reported arginase activity in plasma in clinical settings, which seemingly conflicts with our findings. Roth and co-workers [11,16] have shown that liver transplantation leads to an immediate decrease in plasma arginine to virtually zero with a stoichiometric increase of plasma ornithine, which is highly indicative of arginase activity. We were able to reproduce these findings, although the decrease in arginine levels was not as great as described by Roth et al. [11,16] Moreover, plasma concentrations of other amino acids, such as methionine and alanine, decreased as well, reflecting restoration of liver function. Nonetheless, the specific steep decline of arginine in the first 3 min following reperfusion and the simultaneous increase in ornithine levels is in fact highly indicative of arginase activity. Arginase-1 plasma levels were 100-fold higher after liver transplantation than during liver resection and 1000-fold higher than normal reference values. In vitro assessment of plasma obtained 3 min following transplantation revealed specific and stoichiometric changes in arginine and ornithine concentrations that were related to the arginase-1 concentration. Arginase activity/μg of arginase-1 (specific activity) appeared to decline at increasing arginase-1 concentrations. This is probably due to the high initial reaction velocity at high arginase-1 concentrations, leading to a rapid decline in arginine concentration and arginase activity. In samples with an arginase-1 concentration <1.5 μg/ml, arginine depletion did not occur. At these low enzyme concentrations, there was a linear relationship between plasma arginase-1 concentration and arginase activity. Moreover incubation of plasma samples with 20 mmol/l arginine (above the Km) showed that the intrinsic activity of the enzyme was preserved during incubation. Theoretically, the activity of arginase-1 at low enzyme and/or substrate concentrations may be limited by endogenous arginase inhibition. The most potent endogenous arginase inhibitor known is NOHA (Nω-hydroxy-L-arginine), an intermediate of NO synthesis. However, regarding the high NOHA concentrations required to cause arginase inhibition (IC50≈400 μmol/l) [33], it appears unlikely that any of the presently known arginase inhibitors affected the present results.

Owing to limited availability of methods to quantify arginase (-1 and -2) protein content in biological samples, various semi-quantitative arginase activity assays, relying on supraphysiological pH and arginine concentrations, have been developed [20,34]. The widespread use of such biochemical assays has led to the interchangeable use of the terms arginase activity and arginase concentration. The in vitro experiments in the present study were designed to study arginase activity under physiological conditions, rather than to quantify plasma levels of arginase. The results show that detection of arginase activity in a plasma sample under optimal biochemical conditions does not necessarily imply that arginase is active in plasma under physiological conditions.

Currently, there is growing interest in the role of arginine and arginase in haemolytic anaemia [10,34]. In a previous study [10], it was reported that haemolytic patients with sickle cell anaemia had reduced arginine levels and a 5-fold increase in plasma arginase activity, measured by a semi-quantitative assay. In the same study, it was found that erythrocyte arginase activity was increased similarly. Another study [34] reported a 14-fold increase in erythrocyte arginase activity in sickle cell disease, measured semi-quantitatively. In the light of our present results, which show that a considerable amount of plasma arginine catabolism is performed by arginase-1 within the intact erythrocyte, it can be speculated that increased arginase activity in the blood of sickle cell patients should be ascribed to increased arginase activity in the cellular fraction, rather than to increased arginase levels in plasma.

Finally, it has been suggested that increased circulating arginase concentrations disturb arginine metabolism following blood transfusion due to haemolysis of stored erythrocytes [20]. We found that normal erythrocytes contain 17 μg of arginase-1/ml of cells. Therefore 1 unit of erythrocyte concentrate (300 ml) contains 5.1 mg of arginase-1. Considering a distribution volume of 3 litres (plasma volume) for these erythrocytes after transfusion, 1 unit of packed erythrocytes can increase arginase-1 plasma levels with 1.7 μg/ml in the extreme situation of 100% haemolysis of the transfused erythrocytes. This theoretical example shows that plasma arginase-1 activity during blood transfusion will probably not affect arginine metabolism substantially. This assumption is confirmed further by clinical observations in patients receiving massive blood transfusion during thoracoabdominal aorta surgery, showing moderate changes in plasma arginase-1 concentrations but no changes in plasma arginine concentrations (S. J. P. Hanssen, M. C. G. van de Poll and W. A. Buurman, unpublished work).

In conclusion, increased plasma levels of arginase-1 occurring during liver resection do not lead to arginine breakdown. The threshold beyond which the plasma level of arginase-1 significantly affects plasma arginine concentration is probably rarely reached in clinical practice, with the exceptions of liver transplantation.

Abbreviations

     
  • NOHA

    Nω-hydroxy-L-arginine

  •  
  • SSA

    sulfosalicylic acid

We thank Dr J. Rozing (Department of Biochemistry, Maastricht University, Maastricht, The Netherlands), Dr W. H. Lamers (Department of Anatomy and Embryology, Maastricht University, Maastricht, The Netherlands) and Dr A. J. Meijer (Department of Biochemistry, University of Amsterdam, Amsterdam, The Netherlands) for fruitful discussions. This study was supported by grants from The Netherlands Organization for Health Research and Development to M. C. G. vdP. (920-03-317 AGIKO) and C. H. C. D. (907-00-033 Clinical Fellowship).

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