Simultaneous study of the metabolic turnover and renal excretion of salivary amylase- 125 I and pancreatic amylase- 131 I in the baboon.

The metabolic turnover of salivary and pancreatic amylase was studied in the baboon, an animal with a serum amylase level and renal clearance of amylase similar to man. Purified amylase was electrolytically iodinated. Although iodinated and uniodinated amylase had similar gel filtration, electrophoretic, enzymatic, glycogen precipitation characteristics, the labeled enzyme was cleared less rapidly by the kidney than was the unlabeled material. However, urinary iodinated amylase which had been biologically screened by the kidney had a renal clearance and serum disappearance rate indistinguishable from unlabeled amylase and thus can serve as a tracer in metabolic turnover studies. Administration of a mixture of salivary amylase-(125)I and pancreatic amylase-(131)I made it possible to simultaneously measure the serum disappearance and renal clearance of these two isoenzymes. The metabolic clearance of both isoenzymes was extremely rapid with half-times of about 130 min. This rapid turnover of serum amylase probably accounts for the transient nature of serum amylase elevation which frequently occurs in pancreatitis. Pancreatic amylase-(131)I was consistently cleared more rapidly (mean clearance ratio: 1.8) by the kidney than was salivary amylase-(125)I. This more rapid renal clearance of pancreatic amylase may help to explain the disproportionate elevation of urinary amylase relative to serum amylase observed in pancreatitis.

Human urine deoxyribonuclease II (DNase II) isoenzymes: a novel immunoaffinity purification, biochemical multiplicity, genetic heterogeneity and broad distribution among tissues and body fluids.

Deoxyribonuclease II (DNase II) was purified from the urine of a 48-year-old male (a single individual) using a column chromatography series, including concanavalin A-agarose and an immunoaffinity column utilizing anti-human spleen DNase II antibody, and was then characterized. Based on the catalytic properties of the purified enzyme, we have devised a technique of isoelectric focusing by thin-layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with a specific zymogram method, for investigating the possible molecular heterogeneity of human DNase II. DNase II in urine as well as the purified form was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 5-7. Since sialidase treatment of the urine sample induced simplification of the isoenzyme patterns with diminishment of anodal bands, it was clear that the multiplicity of the enzyme was in part due to differences in the sialic acid content. On screening of DNase II isoenzyme patterns in urine samples from more than 200 Japanese individuals, only the common isoenzyme pattern was observed and no electrophoretic variations were detected. However, genetic studies of urinary enzyme activity and comparative studies on the activity in urine, semen and leukocytes from the same individuals suggest that the enzyme activity level of DNase II may be under genetic control. The enzyme was widely distributed in human tissues and showed high activities in secretory body fluids such as breast milk, saliva, semen and urine, and leukocyte lysates.

Pancreatic and salivary amylase activity in undernourished Colombian children.

Amylase activities were quantitated in secretions of marginally and severely malnourished Colombian children. In young children with a mean age of 21 months, the relative pancreatic and salivary amylase isozyme activities of urine were significantly changed in marginally malnourished children compared to normal children. There was a relative increase in salivary and decrease in pancreatic amylase activity in the undernourished children and total amylase activity was somewhat decreased. Amylase activity in saliva and tears was significantly lower in these malnourished children. Older children who were more severely malnourished had significantly lower amylase activity in their sera and tears. Thus marginal and severe malnutrition affects the production of amylase by the pancreas and salivary glands of young children distinctly. It significantly suppresses amylase activity in tears, saliva, and serum.

An evaluation of the usefulness of amylase isozyme differentiation in patients with hyperamylasemia.

Amylase assays measure total activity without differentiating the relative contributions of pancreatic- and salivary-type amylase isozymes. Since polyacrylamide electrophoresis allows identification of salivary-and pancreatic-type isoxymes and their respective variants, serum and urine specimens from patients with the clinical diagnoses of mumps (4), pancreatitis (16), or undiagnosed hyperamylasemias (5) were compared with specimens from control subjects. Patients with mumps had elevations of salivary-type isozymes, while those with pancreatitis had elevations of pancreatic-type isozymes. Elevation of salivary-type isozymes was identified in the five patients who had undiagnosed hyperamylasemias; among these, the isozymes of two originated in neoplastic ovarian tissue and those of three, probably in the salivary glands. Amylase isozyme differentiation cannot unamibiguously identify the tissue source of hyperamylasemia. However, in patients whose hyperamylasemia is of unknown etiology or who respond atypically to therapy, amylase electrophoresis provides identification of the elevated isozyme type, thus providing the basis for the rational selection of further diagnostic procedures.

Charge selectivity and urine amylase isoenzymes.

The urinary excretions of salivary and pancreatic amylase were studied in 718 type I diabetic patients and 51 control subjects, as part of a multicenter study on diabetic nephropathy in 15 Spanish hospitals. It was found that the urinary ratio of salivary to pancreatic amylase (S/P ratio), that in normal subjects is always below 1, was elevated in 35.4% of diabetic patients, whereas microalbuminuria was present in 19.8%. The prevalence of elevated S/P ratio was also higher than that of microalbuminuria at the first years from the onset of the disease, but the prevalence of microalbuminuria was higher in patients with a long duration of the disease. alpha 1-microglobulin and microalbuminuria paralleled their prevalences during the disease, when measured in a group of patients. Overnight urine samples were obtained on three consecutive weeks from the diabetic patients, and a nested ANOVA analysis showed that the intra-individual variation of the urine parameters measured (albumin, salivary and pancreatic amylase, and beta-NAG) was very small and not statistically significant. All these findings suggest that in type I diabetes mellitus, loss of negative charges of GBM would induce preferential excretion of the anionic salivary amylase over the more cationic pancreatic amylase, and that this phenomenon is more frequent and appears earlier than microalbuminuria. The mechanisms for the increased excretion of salivary amylase and albumin into urine seem to be at least partly different. On the contrary, increase in urinary excretion of albumin and alpha 1-microglobulin in these patients are correlated, suggesting a tubular participation in the mechanisms of production of microalbuminuria.

Measurement of amylase isoenzymes in human sera and urine using a DEAE-cellulose mini-column method.

A DEAE-cellulose mini-column method has been developed which allows for the separation and quantitation in human sera and urine of pancreatic and salivary type isoamylases. Determination of the isoamylases was found to be of value in differentiation of hyperamylasemias due to disorders of the pancreas and parotid gland.

[Urinary elimination of multiple forms of gamma-glutamyltranspeptidase and aminopeptidase (author's transl)]. / Etude des formes d'élimination urinaire de la gamma-glutamyltranspeptidase et de l'aminopeptidase.

Concentrated and dialyzed 24-h urines of healthy persons were separated by 105000 X g ultracentrifugation into a pellet (P105) and a supernatant (S105) fraction. Chromatography of the P105 fraction on Sepharose 4B and 2B revealed that gamma-glutamyltranspeptidase and aminopeptidase activities had a molecular mass of 20 X 10(5) to 40 X 10(6), whereas in the S105 fraction soluble gamma-glutamyltranspeptidase and aminopeptidase had 86000 and 160000, respectively. Triton X-100 solubilization was performed on the P105 fraction and on a human renal cortex 40000 X g pellet, used as a reference. All the activity was recovered in both cases in a single peak of detergent gamma-glutamyltranspeptidase and detergent aminopeptidase eluted by filtration on Ultrogel Ac A22. Apparent molecular mass of Triton X-100 solubilized urinary and renal enzymes were 250000 and 243000 for gamma-glutamyltranspeptidase, and 298000 for both aminopeptidases. Protease solubilized forms were obtained by trypsic digestion of detergent urinary and renal forms. Both gamma-glutamyltranspeptidases were found to have an apparent molecular mass of 86000 on Sephadex G 150, which is identical to the value found for the S105 urinary gamma-glutamyltranspeptidase. The aminopeptidases had 238000 and 232000, which is a higher value than the molecular mass of the S105 urinary aminopeptidase. This letter could be a degraded form of the renal aminopeptidase. These findings suggest that gamma-glutamyltranspeptidase and aminopeptidase in the P105 fraction are similar to native renal enzymes. Evaluation of the P105 fraction enzymatic activities may thus be useful in the diagnosis of tubular damage.

Experimentally induced periodontitis in beagle dogs causes rapid increases in osteoclastic resorption of alveolar bone.

This study was undertaken to observe osteoclast differentiation related to inflammatory progression in aggressive periodontitis induced in beagle dogs by ligature of the gingival sulcus. To monitor osteoclastic activity, we used histochemical methods (staining for tartrate-resistant acid phosphatase [TRAP]) to visualize osteoclasts and their TRAP-positive precursors and biochemical methods (ELISA assay of pyridinium crosslinks) to detect bone matrix degradation products in gingival crevicular fluid (GCF), serum, and urine. For histochemical study, tissue specimens were prepared from 3 adult female beagle dogs induced with experimental periodontitis by silk ligature placement below the gingival margin of mandibular molars ligated for 3, 7, and 21 days. For biochemical study for pyridinoline measurement, the 24 mandibular molars of 4 male beagle dogs were ligated. GCF, urine, and serum were collected at day 0 and at 3, 7, 14, and 21 days after ligation. In the early inflammatory phase of ligature-induced periodontitis (day 3), TRAP+ mononuclear and TRAP+ multinucleated cells were present in the gingival connective tissue, and active bone-resorbing cells were found in excavated lacunae at the alveolar crest, but osteoclasts were not infiltrating the periodontal ligament during this early phase. During later stages of the inflammatory process (7 and 21 days), osteoclasts appeared at both the gingival and ligament side of the alveolar bone. Osteoclastic bone resorption appeared to be more severe on the bone surface at the gingival side than on the bone surface of the periodontal ligament side. Measurement of pyridinoline significantly increased in GCF and urine 3 days after ligation. The results suggested that bone at the crest of the alveolar bone is rapidly resorbed within 3 days of inducing experimental periodontitis.

Changes of serum amylase, its isozyme fractions and amylase-creatinine clearance ratio in dogs with experimentally induced acute pancreatitis.

To investigate the diagnostic application of amylase to canine pancreatic diseases, serum amylase activities, its isozyme fractions and amylase-creatinine clearance ratio (ACCR) were analyzed in normal intact dogs and dogs experimentally induced acute pancreatitis. There was no statistic difference between normal male and female dogs. Amylase specific activities in pancreatic tissue extracts were more than 2,300 times higher than that in serum, and were also higher than those in other tissues; parotid and mandibular salivary glands, lung, heart, liver, spleen, duodenum, jejunum, ileum and kidney. Following the chloroform injection into the pancreatic tissue, WBC increased from 6 to 240 hr and serum glucose significantly increased at 72 and 96 hr, and no urine glucose was detected. BUN as well as serum and urine creatinine showed normal levels. ACCR increased until 96 hr without statistic significance. Serum amylase activities increased significantly after 3 hr and its isozyme was separated into 4 fractions (Amy1-Amy4) in contrast to 3 fractions (Amy2-Amy4) in intact dogs. Since this extra Amy1 seen from 1 hr increasing after 6 hr similarly to other 3 fractions, the evaluation of serum amylase and its isozyme fractions was indicated to be useful for the diagnosis of acute pancreatitis in dogs.

A significant dose-dependent relationship between mercury exposure from dental amalgams and kidney integrity biomarkers: a further assessment of the Casa Pia children's dental amalgam trial.

Dental amalgams are a commonly used dental restorative material. Amalgams are about 50% mercury (Hg), and Hg is known to significantly accumulate in the kidney. It was hypothesized that because Hg accumulates in the proximal tubules (PTs), glutathione-S-transferases (GST)-α (suggestive of kidney damage at the level of PT) would be expected to be more related to Hg exposure than GST-π (suggestive of kidney damage at the level of the distal tubules). Urinary biomarkers of kidney integrity were examined in children of 8-18 years old, with and without dental amalgam fillings, from a completed clinical trial (parent study). Our study determined whether there was a significant dose-dependent correlation between increasing Hg exposure from dental amalgams and GST-α and GST-π as biomarkers of kidney integrity. Overall, the present study, using a different and more sensitive statistical model than the parent study, revealed a statistically significant dose-dependent correlation between cumulative exposure to Hg from dental amalgams and urinary levels of GST-α, after covariate adjustment; where as, a nonsignificant relationship was observed with urinary levels of GST-π. Furthermore, it was observed that urinary GST-α levels increased by about 10% over the 8-year course of the study among individuals with an average exposure to amalgams among the study subjects from the amalgam group, in comparison with study subjects with no exposure to dental amalgams. The results of our study suggest that dental amalgams contribute to ongoing kidney damage at the level of the PTs in a dose-dependent fashion.

Amylase thermolability in body fluids.

The thermolability of amylase was measured in saliva, pancreatic juice, urine, adult and neonatal sera. The mean percentage thermolability from these fluids was 100%, 99%, 87%, 44% and 23% respectively. In patients with acute pancreatitis and mumps the amylase was 84% and 83% thermolabile during the acute phase. On resolution of the pancreatitis this dropped towards normal. Patients with a pancreatic pseudocyst showed a high mean percentage thermolability (82%). These results could suggest that a component of amylase in human serum is not of pancreatic or salivary origin. In addition, this simple technique may be helpful in the diagnosis of pancreatic pseudocyst.

Early changes of urinary amylase isoenzymes in diabetes mellitus.

The altered excretion of isoenzymes of amylase in urine was used as an early indicator of the loss of electric charges in the glomerular basement membrane, in 202 juvenile-onset insulin-dependent diabetic patients, compared with the pattern of excretion in 51 normal subjects matched for age and sex. Diabetics showed an increased excretion of salivary amylase. The salivary to pancreatic amylase ratio in urine (S/P ratio) was always below 1 in control subjects, but was elevated in 33.2% of diabetics, although microalbuminuria was present in only 26.2% of diabetic patients. The concentrations of other proteins in urine were within the reference ranges in nearly all patients, indicating that the kidney was not seriously affected. The increased salivary amylase excretion was not due to changes in the plasma concentration of any of the isoamylases, but to a real increase in excretion, as its fractional excretion in relation to creatinine clearance was clearly increased (1.0 +/- 0.7 vs. 1.52 +/- 1.99, p < 0.05), and the ratio of their clearances was also increased (0.35 +/- 0.18 vs. 0.49 +/- 0.61, p > 0.05). Moreover, the prevalence of altered S/P ratios was higher than the prevalence of microalbuminuria (36.6% vs. 18.8% of patients in the first decade of evolution of insulin-dependent diabetes mellitus). Altered S/P ratios were most prevalent in the first decade, whereas microalbuminuria was most prevalent in the second decade of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal clearance of pancreatic and salivary amylase relative to creatinine clearance in patients with renal disease and proteinuria.

To study the charge-selective properties of the glomerular filter in renal disease, we measured the fractional clearance, relative to creatinine clearance (ECC), of the amylase isoenzymes pancreatic amylase and salivary amylase, which have identical size but different charge. In 63 healthy subjects the mean (and SD) fractional excretion of pancreatic amylase, 4.07% (1.24%), was fourfold that of salivary amylase: 1.02% (0.54%). For 29 patients with renal disease and proteinuria, the mean fractional excretion of pancreatic amylase was significantly lower, 3.31% (1.94%), and that of salivary amylase significantly higher, 2.06% (1.41%), than in controls. In these patients, fractional excretions of both these isoenzymes were negatively correlated with urinary excretion of beta 2-microglobulin and ECC. Evidently, differences in clearances of pancreatic and salivary amylase are a consequence of differences in charge-related glomerular filtration. The relative increase of salivary amylase clearance in patients with renal disease and proteinuria is most probably caused by a loss of the charge-selective properties of the glomerular basement membrane.

Differential assay of salivary and pancreatic alpha-amylase in serum and urine, with use of monoclonal antibody to human salivary amylase immobilized on bacterial cell wall.

We previously reported (Clin Chim Acta 1986;159:89) that bacterial cell wall chemically coated with a monoclonal antibody specific to human salivary (S) amylase (EC 3.2.1.1) could be successfully used to separate S and pancreatic (P) amylase in solution. We have now applied this method to serum and urine samples and found that the activities of S and P amylases so measured correlated well with those measured by the isoamylase inhibitor method. The present method is simple and reliable for routine clinical tests.

Urinary amylase isoenzymes and amylase polymorphism variants in families with diabetes mellitus type 1.

Insulin-dependent diabetes mellitus type 1 is an autoimmune disease of pancreatic beta-cells with a certain genetic predisposition that is not yet clear. In spite of the confirmed association of diabetes mellitus type 1 with several HLA haplotypes it is considered that other loci must be involved for total genetic susceptibility to the disease. The relationship of insulin deficiency and decreased pancreatic amylase activity suggests that insulin itself is a direct activator of amylase gene expression. Endocrine pancreatic function was monitored by the indirect non-invasive method of urinary pancreatic amylase activity determination (expressed in percentage of total amylase activity) in diabetic children, their parents, healthy sisters and brothers, and in a separate group of women with diabetes type 1 of over 20 years duration. The incidence of hereditary amylase polymorphism variants in these subjects was also ascertained. Decreased pancreatic amylase activity in urine (under 58%) was found to be a characteristic trait in diabetics, and a susceptibility trait in asymptomatic family members. Normal pancreatic amylase activity (66.7 +/- 5.4%) is rare in diabetic patients type 1, but may be seen as a favourable prognostic trait, representing resistance to diabetic complications. The results support the suggestion that hereditary predisposition to the disease is inherited from the father rather than the mother, and that heterozygous amylase polymorphism variants protect their carriers against diabetes mellitus type 1.