Holger Skoldefors

Pancreas-Specific Protein New Serum Marker for Graft Rejection in Pancreas-Transplant Recipients

A radioimmunoassay for a novel human pancreatic protein (pancreas-specific protein, PASP) has been developed. We studied the possibility that serum PASP levels reflect pancreas-graft rejections in human pancreas-transplant recipients. Ten patients subjected to combined pancreas-kidney transplantation and 4 patients subjected to pancreas transplantation alone were studied. Twelve kidney recipients served as control subjects. On several occasions, PASP levels were elevated at kidney rejections in patients with combined pancreas-kidney grafts and then decreased after antirejection therapy, although no other indications for concomitant pancreas-graft rejection were at hand. In the recipients of pancreas grafts alone, PASP levels increased before or at the same time as graft rejections were indicated by current methods. In two cases of chronic graft rejection, PASP rose to high levels long before hyperglycemie occurred. In the control group of kidney-graft recipients, PASP levels were stable and were not affected by high serum creatinine levels, kidneyrejection episodes, or antirejection therapy. This study indicates that PASP may be a good serum marker for pancreas-graft rejection.

A novel assay for pancreatic cellular damage. IV: Serum concentrations of pancreas-specific protein (PASP) in acute pancreatitis and other abdominal diseases

Pancreas-specific protein (PASP) was compared with serum amylase in 95 episodes of acute pancreatitis with the diagnoses supported by elevated amylase levels. The etiology was typical for Scandinavian countries, with alcohol as the predominant factor, followed by cholelithiasis. PASP values were clearly raised in all patients, except in three cases found to have high salivary-type amylase levels, and one patient with recurrent alcohol pancreatitis. The rise of PASP levels were in general more pronounced than the corresponding amylase elevations, especially in severe pancreatitis. The elevations were generally parallel for the two analytes, but in 41% of the cases PASP levels remained elevated 2–11 days longer than the corresponding amylase levels. PASP was, however, eliminated from the circulation at a rate comparable to that of amylase. The serum range of PASP for 259 healthy subjects was 15–111 μg/L with 95% of the values within 16–98 μg/L. The upper reference level was set at 100 kg/L. PASP levels were also determined for 291 patients with disorders other than acute pancreatitis. Serum levels in patients with renal insufficiency (n = 12), primary biliary cirrhosis (n = 9), and diabetes mellitus (n = 17) were equal to those in healthy subjects. Eight patients of 173 with acute abdominal disorders and no evidence of pancreatitis had elevated PASP levels as well as 4 patients with prostatic carcinoma (n = 28) and 2 patients with benign prostatic hyperplasia (n = 16). PASP values were low in chronic painful pancreatitis (n = 15) and pancreatic cancer (n = 11).

Novel assay for pancreatic cellular damage: 1. Characterization of protein profiles in human pancreatic cytosol and purification and characterization of a pancreatic specific protein.

The protein patterns of cytosols from normal human pancreas and pancreatic carcinoma were studied by a two-dimensional separation technique using high-performance liquid chromatography followed by isoelectric focusing on polyacrylamide gels and visualization of the focused proteins by Coomassie Blue staining. Almost identical protein patterns were obtained for 20 different specimens from normal pancreas, whereas quite different protein patterns were found in 12 samples of pancreatic carcinoma. A major protein in normal pancreatic cytosol, not identical to any macromolecule previously tested as a marker for pancreatic function, was selected for further studies. The protein was not found in specimens of pancreatic carcinoma. It was purified by a single step chromatofocusing procedure, focused at pH 6.9, and moved as one single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 44,500 daltons. Total amino acid analysis revealed a high concentration of glutamic acid, leucine, and lysine. The purified protein had no amylase activity or lipase immunoactivity. It constituted ∼2% of the total normal pancreatic cytosol protein. Later immunological studies have shown the protein to be highly specific for normal human pancreas, indicating a possible future use as a marker for pancreatic cell damage.

Further characterization of the estrogen binding macromolecule in human pancreas

The estrogen binding protein in human pancreas has been purified from pancreatic cytosol by chromatography on Concanavalin-A-Sepharose and hydroxyl-apatite followed by ion-exchange chromatography carried out using a fast-protein liquid chromatography apparatus (FPLC). The purified protein, still able to bind labelled [3H]estradiol, appeared as one single band corresponding to 31 K in SDS-gel electrophoresis. Total amino acid analysis revealed high levels of histidine, glutamic acid and leucine. The capacity of the purified protein to bind estrogens could be increased more than 4-fold by addition of a cytosolic factor, probably being a small peptide, that is present in crude cytosol, but lost during the purification procedure. The iodinated protein does not bind to DNA-cellulose or phosphocellulose, and shows no similarities to estrogen receptor proteins.

A novel assay for pancreatic cellular damage. III: Use of a pancreas-specific protein as a marker of pancreatic graft dysfunction in humans

Pancreas-specific protein (PASP) is a recently isolated and partially characterized major protein in the human pancreas. It has not been described previously. Serum levels of PASP and amylase were analyzed in 21 patients subjected to combined renal and segmental pancreatic transplantation with both organs obtained from the same donor and in eight kidney transplant patients. In the pancreas transplant patients, PASP and amylase levels were elevated in episodes of graft pancreatitis. With chronic graft rejection, PASP rose to high levels long before other indications. In episodes of renal rejection, the levels of PASP, but not always of amylase, were elevated on several occasions. They decreased after antirejection therapy. This may indicate accompanying pancreatic graft rejection. PASP and amylase levels were stable in kidney transplant patients and were not affected by serum creatinine levels, renal rejection, or antirejection therapy. The results support earlier observations that renal rejection in combined pancreas and renal transplant patients may or may not be accompanied by a rejection process in the pancreatic graft. PASP may be the means by which to tell when the pancreatic graft is involved.

A novel serum assay for pancreatic cellular damage. II: High tissue specificity of a pancreatic protein

A previously unknown major protein in human pancreatic cytosol has been purified and partly characterized. The protein, designated pancreas specific protein (PASP), has a molecular weight of 44,500 and a PI of 6.9. A two-dimensional gel separation technique revealed the protein to be specific for normal pancreatic tissue. Antibodies against PASP were raised in rabbits and a radioimmunoassay was developed for the quantitation of this protein. The following concentrations of PASP (mg/kg wet weight) were found in human tissues: normal pancreas 100–1 1,000; pancreatic carcinoma 0.1–20; prostate 0.5–5; and 13 other tissues <0.5. The levels of PASP in peripheral serum were <0.1 mg/L in normal subjects, 0.7–3 mg/L in cases of acute pancreatitis, and <0. 1 mg/L in cases of pancreatic carcinoma, prostatic diseases, and other abdominal diseases investigated. The high tissue specificity and the specific elevation of serum PASP levels in acute pancreatitis may indicate a use of this protein as a marker of this pancreatic condition.

The estrogen binding protein in human pancreas: Concentrations in subcellular fractions of normal pancreatic tissue, in duodenal juice during pancreatic stimulation and in peripheral serum in normal and pathological conditions

The steroid binding properties of the human pancreatic estrogen binding protein (hEBP) in cytosol were studied by equilibrium dialysis. A high ligand specificity of the protein was revealed. hEBP in cytosol binds unconjugated steroid estrogens with a medium affinity (Kd = 10(-7) M) but does not bind conjugated estrogens or unconjugated androgens, gestagens, glucocorticoids or cholesterol. Quantitation of hEBP by radioimmunoassay in subcellular fractions of human pancreatic tissue indicated that the protein is translocated into different subcellular compartments. Duodenal juice taken from patients following stimulation of pancreatic secretion by food ingestion (Lunds test) showed high hEBP concentrations, and the levels of hEBP changed concomitantly with the levels of pancreatic isoamylase, indicating that hEBP secretion was stimulated by food ingestion. The levels of hEBP in peripheral serum from healthy subjects showed no sex difference, but were positively correlated to age. Highly elevated (10-20-fold) hEBP levels were found in serum from patients with acute pancreatitis, while normal serum hEBP values were found in other abdominal diseases. It is speculated that hEBP might have a specific role in the transport of estrogens from the peripheral circulation via the pancreas to the duodenum. The elevated hEBP levels in patients with acute pancreatitis indicate that this protein may be used as a marker of cellular damage in the pancreas.

Aging and urinary oestrogen excretion in the male.

Conflicting results have been presented about the influence of aging upon the urinary excretion and the blood levels of oestrogens in the male. While Behrsohn & Oelefse (2) found an increased urinary excretion of conjugated oestrone, oestradiol-17p and oestriol with increasing age, Pincus (17, 18) found a slight continuous decrease in the excretion of oestrone + oestradiol-17p and an almost constant excretion of oestriol. Kaufmann (13) noted no agedependent change in the excretion of oestrone and oestradiol-17p. The studies on blood hitherto reported indicate increased levels of unconjugated oestrone and oestradiol-17p with increasing age (8, 16,20). We have studied the influence of aging upon the urinary excretion of low polar oestrogens (oestrone + oestradiol-17p) in normal healthy males aged 20-79 years. The subjects below 50 years of age had proven fertility or normal spermiograms. The older subjects had been hospitalized for minor operations. There were no indications for endocrinological perturbations or abnormalities in the prostate. Values for haemoglobin, Na+, K+, sedimentation rate, urinary residual nitrogen and urinary sediment were normal and all patients were free from medications. 24 hour urine samples were collected preoperatively. Low polar oestrogens were determined according to Carlstrom and Furuhjelm (6).

Influence of Aging Upon the Urinary Hormone Excretion in the Male

Abstract. The urinary excretion of LH, low polar oestrogens, neutral C12 and C21 steroids was measured in normal healthy males aged 20–79 years. No significant changes could be noted for the excretion of LH, pregnanediol and 17‐ketogenic steroids between 50 and 79 years. The excretion of androsterone and aeticholanolone was significantly lower in the oldest group (70–79 years). A significant drop in the excretion of DHA was noted at approx. 60 years. The excretion of low polar oestrogens remained constant from 20 to 55–59 years, but showed a highly significant (P >0.001) decrease at about 60 years. This drop might reflect a sudden decrease in the plasma levels of oestrone sulphate due to a decreased sulphurylating activity in the liver.

Analysis of an estrogen binding macromolecule in human pancreas by radioimmunoassay

A radioimmunoassay for quantitation of the human pancreatic estrogen binding protein (hEBP) was developed using polyclonal rabbit hEBP antiserum and iodinated purified hEBP. Parallel dose-response curves were obtained when serial dilutions of human serum and of cytosols obtained from human pancreas, prostate and colon were analyzed simultaneously with serial dilutions of purified hEBP standard. Very high levels of hEBP (500-1000 mg/kg wet weight) were found in normal pancreas. High as well as medium levels were found in pancreatic carcinoma tissue and medium values (0.1-1 mg/kg wet weight) in prostate, colon and ovarian tissue. Other tissues and serum from healthy volunteers showed low values, usually below 0.1 mg/kg. When serial dilutions of rat pancreatic cytosol were analyzed in the hEBP assay, [125I]hEBP was displaced by the rat preparation, but the dose-response curves were not parallel to the standard curves, indicating similarity but non-identity between the estrogen binding proteins in human and in rat pancreas.