Serum procarboxypeptidase A and carboxypeptidase A levels in pancreatıc disease

Introduction

Procarboxypeptidase A (pro-CPA) is the zymogen of an exopeptidase, carboxypeptidase A, which discharges from peptides and proteins carboxyl terminal amino acids whose side chains comply to specific requirements of this enzyme. The active enzyme can be isolated from activated beef pancreatic juice drippings of fresh pancreas glands; the enzyme can be isolated from acetone powder of unactivated bovine pancreas glands. ¹

Carboxypeptidase A (CPA, EC 3.4.17.1) is a much studied and described metalloprotease, and thus coursing as a prototypic enzyme for many metalloenzymes involving physiologically specific proteases such as angiotensin-converting enzyme and enkephalinase.^2,3

CPA is created by the pancreas ⁴ and it thus met the canon of tissue privy to primitive markers. With the development of specific and sensitive assays for CPA and its precursor pro-CPA, it becomes probable to endorse baseline values for these two proteins in a healthy adult.^5,6

Serum pro-CPA and CPA levels were prevailed by the obstructed pancreatic duct. Observed pancreatic inadequacy that is often marked with the attending diarrhea, diabetes and frequency associated with pancreatic diseases. However, the clinical values of these markers for the diagnosis of inflammatory diseases, acute pancreatitis and pancreatic cancer are very important.

We suggested that in pancreatic disease, the changes in serum pro-CPA and CPA result from the pancreas gland. In this study, we aimed to determine the serum levels of pro-CPA and CPA in patients with pancreatitis and pancreatic cancer. We compared these levels with those of healthy controls and among each pancreatic disease.

Materials and methods

Study groups comprised 96 cases with acute pancreatitis (male/female 49/47), 101 chronic pancreatitis patients (male/female 48/53) and 98 pancreatic cancer cases (male/female 50/48). The study protocol was approved by the medical Ethics Committee of Haseki Training Hospital, Istanbul, and was in accordance with ethical standards formulated in the Helsinki Declaration of 1975. Informed consent was obtained from all the participants.

Patients in the acute pancreatitis group had signs and symptoms of pancreatitis accompanied by elevations in biochemical tests (i.e. amylase, lipase). Diagnosis of chronic pancreatitis was confirmed with one or usually a combination of radiological tools (i.e., ultrasonography, computerized tomography and endoscopic ultrasonography). A diagnosis of pancreatic cancer was usually made by a combination of biochemical (tumor markers), radiological and pathological tests and surgical exploration. The number of cancer patients by stage is as follows: stage 1, 20; stage 2, 25; stage 3, 33; and stage 4, 20. No patient had anticancer treatment previously. No patients had tuberculosis and any other accompanying diseases that could influence the pro-CPA and CPA levels. A total of 96 healthy participants (male/female 49/47) who were admitted to the hospital for a checkup were included as a control group.

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilurubin, direct bilirubin, albumin and carbohydrate antigen (CA) 19-9 levels were measuredspectrophotometrically by the investigators with Olympus AU2700 autoanalyzer. Serum amylase and lipase concentrations were measured using an enzymatic assay (Boegringer Mannheim Systems, Germany). Serum pro-CPA and CPA activities were measured by investigators blinded to the patient’s diagnosis by the methodology described previously.^5,6 Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid)], dl-benzylsuccinic acid, clostripain and dithiothreitol (Cleland’s reagent) were purchased from Sigma (MN, USA). Semimicro cuvettes were purchased from Fisher. The substrate N-acetyl-phenylalanyl-3-thioaphenylalanine (NAcPSP) was obtained from Peptides International (MN, USA). CPA activity was determined by adding serum samples to Ellman’s reagent and incubated for 5 min at 22°C to allow free sulfhydryl groups in the serum to react to completion. NAcPSP was then added and the mixture was immediately dispensed in equal volumes into each of the three plastic cuvettes with 1-cm path lengths. One of these cuvettes (the blank) contained 1 μl of a CPA inhibitor, 0.4 M DL-benzylsuccinic acid, and the other two cuvettes (the ‘tests’) each contained 1 μl of water. An initial absorbance was recorded and the cuvettes were incubated at 37°C. Absorbance was monitored at 412 nm every hour or hour and a half for a total of 3 h with a Zeiss PM6 spectrophotometer. After 3 h, the absorbance was measured using one common cuvette to ensure the precision of the cuvettes. Activation of pro-CPA in the serum samples was achieved with clostripain. Serum (0.040 ml) was combined with 0.020 ml of clostripain (500 units/ml) and incubated at 37°C for 5 min, after which 1.440 ml Ellman’s reagent and 1.020 ml H₂O were added. After incubating for 5 min at room temperature, the assay proceeded as previously outlined for CPA with the addition of NAcPSP. Serum pro-CPA and CAP activities are measured in U/L.

Statistical analysis

All data were analyzed in terms of mean or mean ± standard deviation (SD). Comparisons between groups were made using analysis of variance (ANOVA). Post hoc significances in the ANOVA were assessed by the ‘TAMHANE’ test (because of not equal variances in groups). p Values of <0.05 were considered statistically significant.

Results

The median age of patients with acute pancreatitis, chronic pancreatitis, pancreatic cancer and control group was 49 (29–70), 52 (21–78), 56 (28–82) and 48 (25–73) years, respectively. Biochemical tests are given in Table 1 . Levels of serum pro-CPA and CPA are expressed as the mean ± SD, range and p values, in Table 2 and Figure 1 .

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Table 1.

Clinical variables and laboratory findings of the cases (mean values)

Variables	Acute pancreatitis	Chronic pancreatitis	Pancreas cancer	Control group
N	96	101	98	96
M/F	49/47	48/53	50/48	48/48
Years	49 (29–70)	52 (21–78)	56 (28-82)	48 (25–73)
Biochemical parameters
Hb (g/dl)	11.35	11.22	11.20	12.88
WBC (mm3)	12.959	8.027	7.923	7.730
PLT (mm3)	243.312	275.673	283.448	282.895
AST (U/L)	246.09	24.19	96.93	25.08
ALT (U/L)	251.10	21.24	107.12	20.45
GGT (U/L)	294.27	110.76	289.20	44.04
ALP (U/L)	385.32	361.35	795.56	227.0
Tbilrubin (mg/dl)	2.96	0.88	11.17	1.21
Dbilrubin (mg/dl)	1.94	0.55	7.77	0.75
Albumin (g/dl)	3.14	3.97	5.93	4.02
Amylase (U/L)	8133.54	77.91	240.70	57.77
Lipase (U/L)	776.90	40.70	246.41	41.76
CA 19.9 (U/ml)	–	58.10	440.70	–

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Table 2.

Comparison of serum procarboxypeptidase A and carboxypeptidase A levels in cases with pancreatic diseases

Study groups	Serum procarboxypeptidase A (U/L)	Serum carboxypeptidase A (U/L)
CG	2.12 ± 0.28 (1.5–2.8)	0.09 ± 1.65 (0.02–1.97)
AP	17.23 ± 1.93 (12.5–20)	0.61 ± 0.21 (0.15–1)
CP	4.04 ± 1.13 (2.0–6.9)	0.21 ± 0.6 (0.1–0.41)
PC	39.02 ± 14.78 (12.8–79.4)	1.63 ± 0.41 (1–3)
p Values
AP-CP	0.001	0.634
AP-CG	0.001	0.001
AP-PC	0.001	0. 001
CP-PC	0.001	0.001
CP-CG	0.05	0.05
PC-CG	0.001	0.001

CG: control group, AP: acute pancreatitis, CP: chronic pancreatitis, PC: pancreas cancer.

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Figure 1.

Serum procarboxypeptidase A (pro-CPA) and carboxypeptidase A (CPA) levels in control groups and all patients.

In patients with acute pancreatitis, the serum pro-CPA levels were 17.23 ± 1.93 (range, 12.5–20) and the serum CPA levels were 0.61 ± 0.21 (range, 0.15–1.0). In patients with chronic pancretitis, the serum pro-CPA levels were 4.04 ± 1.13 (range, 2.0–6.9) and the serum CPA levels were 0.21 ± 0.6 (range, 0.1–0.41). In patients with pancreatic cancer, the serum pro-CPA levels 39.02 ± 14.78 (range, 12.8–79.4) and the serum CPA levels were 1.63 ± 0.41 (range, 1.0–3.0). In the control group, the serum pro-CPA levels were 2.12 ± 0.28 (range, 1.5–2.8) and the serum CPA levels were 0.09 ± 1.65 (range, 0.02–1.97).

Serum pro-CPA and CPA levels were significantly higher in acute and chronic pancreatic cancer patients compared to the control group (p < 0.001). Pancreatic cancer patients had significantly higher serum pro-CPA and CPA levels when compared with acute and chronic pancreatitis cases (p < 0.001). Serum pro-CPA and CPA levels were higher in acute pancreatitis compared to the chronic pancreatitis (p < 0.001). These parameters were lower in patients with chronic pancreatitis and control groups (p < 0.001).

Discussion

In this sudy, we demonstrated that serum pro-CPA and CPA levels were increased in either pancreatitis or pancreatic malignancy.

Acute pancreatitis induces a strong sterile inflammatory response in experimental animal models and in humans. Inflammatory mediators seem to play an important role in the pathogenesis of pancreatitis and inflammatory response.^7–9

The spread of chronic inflammatory molecules by these cells and the deposition of collagen are due to the stimulation of the pancreatic stellate cell. ¹⁰ However, the clinical values of these markers for the diagnosis of inflammatory diseases, acute pancreatitis and pancreatic cancer are very important.

Pro-CPA and CPA produced by the pancreas and their respective serum concentrations are affected by pathologic process in the gland. In a study it was demostrated that the ductal cells of the gland should have enhanced serum concentrations of pro-CPA, a protein that is made in the acinar cells. ¹¹

We suggested that with the progress of the pancreatic diseases, the pancreatic duct gets obstructed, resulting in the damage of the upstream acinar cells and release of pro-CPA. It would be supposed that in a healthy individual in whom a pancreatic disease had originated, there would be enhancing serum levels of pro-CPA, as more and more acinar cells were affected by the obstructed pancreatic duct.

The presence of these markers is often associated with pancreatic disease. With respect to CPA, in the absence of inflammation as in pancreatitis, there would be no mechanism for the activation of the pro-CPA. Therefore, these values would remain within the regular range until the loss of the functioning acinar cells would engender an elevated refusal in the serum content of the active enzyme. ¹² Our results were consistent with that of Shamamian and Matsugi and collegaues, in that we found higher serum pro-CPA and CAP levels in patients with pancreatic cancer.^11,12

Our study demonstrated that pro-CPA and CPA levels will elicit the best measure of pancreatic diseases, especially pancreatic cancer development.

This study showed an important correlation between serum pro-CPA, CPA levels and pacreatic diseases especially pancreatic cancer. But, we did not show the correlation between serum levels and tumor location, metastasis and the size of the tumor.

In conclusion, pro-CPA and CPA levels may play an important role in the inflammatory disease of the pancreas. Furthermore, these parameters may be a serum marker for the diagnosis of pancreatic diseases.