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OBJECTIVE--Increased urinary, excretions of several plasma proteins with different molecular radii RESEARCH DESIGN AND METHODS--Urinary excretion rates of proteins mentioned above in timed overnight urine samples were evaluated in 61 [...]

Microalbuminuria is generally considered as the best available noninvasive predictor of diabetic nephropathy. However, several studies have shown that increases in certain urinary proteins (immunoglobin G [IgG] [1-3], transferrin [Tf] [...]

Increased urinary excretion of certain plasma proteins, such as immunoglobulin G (IgG), ceruloplasmin and transferrin, with different molecular radii of 55Å or less and different isoelectric points have been reported to precede development of microalbuminuria in patients who have diabetes mellitus with hypertension. We examined how hypertension affects these urinary proteins in a diabetic state.

Background/Aims:To examine whether chronic intake of vegetable protein added to animal protein diet affects renal hemodynamics or not, we studied effects of three kinds of diets containing various amounts of animal and vegetable protein with 1-week dietary program in each on renal hemodynamics. Methods:The crossover design of different amounts of vegetable protein added to the constant amount of animal protein was applied to two groups of 7 healthy individuals after the control dietary program. Renal function and 24 hours’ urinary albumin excretion rate (AER) were examined on every 7th day of three consecutive 1-week dietary programs. Results:Glomerular filtration rate (GFR; sodium thiosulphate clearance) and renal plasma flow (RPF) significantly decreased after decreasing the intake of animal protein by one third with keeping the amount of vegetable protein constant. The results when substituting vegetable protein for some of the animal protein in the diet without changing the total amount of protein were identical. The filtration fraction and AER did not change over the study periods regardless of dietary composition. Conclusion:The lack of an effect a 1-week intake of vegetable protein added to animal protein on GFR and RPF suggests that vegetable protein may be excluded from lists of restriction in low protein diet therapy in patients with renal insufficiency.

Background/Aims:The aim of this study was to examine the relationship between the degree of urinary copper excretion and stages of diabetic nephropathy. Methods:Copper, ceruloplasmin and albumin concentrations were measured in serum and urine samples from 41 type 2 diabetic outpatients with different stages of nephropathy and from 10 healthy controls. The copper/albumin and copper/ceruloplasmin ratios in serum and urine were determined. Furthermore, we examined whether free copper ions are dissociated from ceruloplasmin under various pH conditions. Results:Urinary copper concentrations significantly increased only in macroalbuminuric patients. The copper/ceruloplasmin and copper/albumin ratios in urine were consistently greater than those in serum which were not different between patients and healthy controls except the copper/albumin ratio in macroalbuminuric patients. The ratios in urine decreased in parallel with the progression of nephropathy. Copper was found to be released from ceruloplasmin under acidic conditions. Conclusion:Urinary copper excretion in healthy controls may be the result of dissociation from the albumin-copper complex of serum during its passage through the kidney. In diabetic patients with advanced nephropathy, urinary copper excretion may be due to dissociations from both copper-albumin and ceruloplasmin-copper complexes filtered through the damaged glomerulus. Overloading of urinary copper to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy.

To examine whether hemodynamic changes in response to acute protein loadings with different protein sources cause increases in urinary excretion of plasma proteins in healthy subjects, urinary excretions of various plasma proteins with various molecular radii and isoelectric points, namely albumin (Alb), IgG, IgG4, ceruloplasmin (CRL), and α2-macroglobulin (A2), were measured in healthy subjects after ingestion of a beef meal or of a tuna fish meal. Significant increases in urinary excretions of the negatively charged IgG4 and CRL and of the neutrally charged IgG were found in parallel with enhanced creatinine clearances after each protein ingestion. These renal responses returned to basal levels 9 h after the test. This finding suggests that in healthy subjects, the increase in glomerular filtration rate after acute protein loading caused selective enhancement of urinary excretions of plasma proteins with a molecular radius of approximately 55 Å (the radius of IgG, IgG4, and CRL), irrespective of the charge barrier of the glomerulus. The increases in these three plasma proteins may be induced by leakage via the shunt pathway in the glomerulus, as proposed earlier (see text). In contrast, increases in urinary excretions of A2 and Alb were not found. The former finding may be explained by the possibility that A2 would not pass through this pathway, since the molecular radius of A2 (88 Å) is larger than that of IgG, although the latter finding may be partially explained by preferential renal tubular reabsorption of Alb.

The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hvperglycemia hut also genetic factors may contribute to the development of diabetic nephropathy. Heparan sulfate proteoglycan (HSPG) is thought to play an important role as a component of the charge selectivity barrier in the glomerular basement membrane. Recently, a BamHI restriction fragment length polymorphism (RFLP) in the HSPG gene (HSPG 2) was reported to be associated with diabetic nephropathy in Caucasian insulin-dependent diabetes niellitus (IDDM). The aim of the present study was to examine the contribution of the BamHI HSPG2 polymorphism to the development of diabetic nephropathy in Japanese non-insulin-dependent diabetes mellitus (NIDDM). For this purpose, we recruited 102 patients with diabetic nephropathy and 64 age-matched patients without diabetic nephropathy from Japanese NIDDM patients. Since all the subjects had proliferative diabetic retinopathy, it seems likely that they would be exposed to hyperglycemia for a long time. In the present study, the BamHI HSPG2 genotype and allele frequencies were not significantly different between the patients with nephropathy and the patients without nephropathy. Therefore, we conclude that the BamHI HSPG2 polymorphism is not associated with the development of diabetic nephropathy in Japanese NIDDM.

In two medullary carcinomas of the thyroid gland two types of secretory granules were found electron microscopically in the cytoplasm of the tumour cells. The sizes of the granules in one case ranged 103–345 nm in diameter; they were round in shape, and they co-existed in the same tumour cell. They could not, therefore, be distinctively subdivided into two types. In another case, secretory granules in the cytoplasm closely resemble EC granule in morphology. Using the protein A-colloidal gold (PAG) technique the content of secretory granules could be identified as calcitonin irrespective of their sizes or morphology. Immunoreactivity at the ultrastructural level was fairly well preserved even in the osmium-fixed tumour cells. The labelling index, expressed as a mean number of gold particles per unit square area of the secretory granule, was higher in the non-osmium-fixed tumour cells than in the osmium-fixed. Non-osmium-fixed tumour cells embedded either in epoxy or methacryl resin were almost equally labelled with gold particles. The result indicates that the PAG method is practicable to demonstrate the ultrastructural localization of calcitonin even in the osmium-fixed, epoxy resin embedded material.

Abstract: We studied the effect of surgery on plasma glucagon (IRG) levels in 40 patients divided into 3 groups according to the severity of surgical stress. In the major and moderate stress groups, 30–100% IRG increases were noted one hour after the start of surgery. In these groups, IRG was increased in the 1st to 7th postoperative days and there was a correlation between the amount of increase and the severity of operative stress. In the minor stress group, no postoperative IRG increase was noted. Our results indicate that the severity of operative stress had a direct bearing on the amount of intra- and postoperative IRG increase. We also discuss the origin of hyperglucagonemia in surgical patients.

Summary: Based on the assumption that somatostatin may inhibit peptide release through junctional complexes or through local circulation, an immunofluorescent technique for somatostatin and GLI in the gut was applied in order to investigate whether suppression of GLI release by i.v. administration of somatostatin was a physiological effect of somatostatin or not. Somatostatin-immunoreactive cells (GIF-cells) in the human and canine intestine had no direct cellular contacts with GLI-immunoreactive cells (GLI-cells). This finding suggests that somatostatin in the intestine does not inhibit GLI release through junctional complexes between GIF- and GLI-cells. As to the local circulation, most of GIF-cells in the canine intestine were distributed in the deeper portion of the intestinal gland which corresponds to the upstream sides of the local blood supply of the intestinal gland, as reported byReynold et al. The ratio of GIF-cells to total cells (GIF-cells + GLI-cells) was 68% in the duodenum and 25% in the ileum. In contrast, a limited number of GIF-cells was found in the human duodenum where a few GLI-cells were distributed and a few GIF-cells were seen in the human ileum where a large number of GLI-cells were located. Findings in the dog suggest the possibility that somatostatin inhibits GLI release from GLI-cells through the local circulation system of intestinal glands. However, findings in humans suggest that the same possibility does not apply to the human gut. Differences of population density of intestinal GIF-cells between humans and dogs indicate that the functional meaning of GIF-cells may vary from one species to another.

Abstract: Wolfram syndrome (MIM 222300) is characterized by juvenile-onset diabetes mellitus and optic atrophy. Previous linkage analyses in the United States and UK families have indicated that the gene for Wolfram syndrome (WFS) is localized on the short arm of chromosome 4. We herein confirm the linkage of the WFS locus to D4S3023 on 4p with a two-point LOD score of 3.42 in a large Japanese family with Wolfram syndrome. Multipoint linkage analysis revealed the maximum LOD score of 4.82 between D4S3023 and D4S394. We also evaluated putative health risks in carriers by multiple logistic analysis with independent variables, age, gender, and numbers of affected haplotypes and with dependent variables, such as hearing loss, diabetes mellitus, polyuria, incontinence, psychological illness, and visual acuity. The results showed that the putative disease haplotype increased a risk of hearing loss (odds ratio =35.68, 95% confidence interval =4.12–308.95) and diabetes mellitus (odds ratio =7.57, 95% confidence interval =2.03–28.23) independently. This is the first report of an increased health risk of illness in carriers, other than for psychiatric disease.

The big ACTH fractions available from human plasma and pituitary glands and from porcine pituitary glands were physico-chemically characterized by gel filtration, disc electrophoresis and isoelectric separation.In the case of healthy human subjects, big ACTH fractions were isolated by gel filtration from plasma samples taken during states of acute ACTH hypersecretion such as the lysine-8-vasopressin, insulin or metopyrone tests though none of these fractions were isolated from plasma sampled under normal conditions. Even with no stimulation of ACTH secretion, patients with Cushing's disease gave plasma samples that contained an isolable big ACTH fraction, but such a fraction was hardly isolated from plasma taken from patient with Addison's disease. Both human pituitaries and porcine pituitaries contained an isolable big ACTH fraction. By a gel filtration analysis the molecular weight of the big ACTH was estimated to be higher than 20 000. Disc electrophoresis with an acrylamide gel indicated that big ACTH is strongly basic while small ACTH is more acidic than pH 8.3. Isoelectric separation revealed that the isoelectric point of human big ACTH is higher than pH 10.0 while that of small ACTH is about pH 6.8.

Abstract. An immunohistochemical study using antisera against proline rich salivary peptide P-C and insulin, glucagon, somatostatin and pancreatic polypeptide antisera was carried out on the foetal pancreas at different stages and on the newborn infant's, infant's, child's and adult pancreas to examine the time at which salivary peptide P-C like immunoreactivity appeared in the human pancreas. Salivary peptide P-C like immunoreactive cells first appeared as a few scattered cells in the foetal pancreas after 16 weeks of gestation and gradually increased in numbers during gestation. The cells corresponded only to insulin immunoreactive cells in the foetal, newborn infant's, infant's, child's and adult pancreas. Only some of the insulin immunoreactive cells in the foetal pancreas contained salivary peptide P-C like immunoreactivity while the majority of those in the infant's pancreas and all those in the child's and adult pancreas did so. The findings, together with the fact that the full sequence of salivary peptide P-C is identical to the COOH-terminal 44 amino acid residues of Salivary Protein C, led to the possibility that peptide P-C like immunoreactivity in the human pancreatic B-cells was not a moiety of the precursor of insulin and pro-insulin, but a moiety of Salivary Protein C. It has been suggested that, in saliva, Salivary Protein C aids in maintenance of the calcium concentration. Based on the hypothesis that peptide P-C like immunoreactivity in the human pancreatic B-cells may play some role in insulin release through the maintenance of the calcium concentration, the present finding seems to explain the fact that the mechanism for insulin release in the foetal pancreas is immature in spite of sufficient biosynthesis of insulin.

Abstract. Antisera against proline rich peptide P-C, recently isolated from human whole saliva were raised in rabbits by injections of peptide P-C-BSA conjugates. Immunohistochemical study using the antisera was carried out on human salivary glands, gut and pancreas. The results showed that peptide P-C like immunoreactivity was present not only in the salivary glands but also in the pancreatic islets, though not in the gut. Furthermore, immunostaining of adjacent thin sections revealed that cells reacting with antisera against peptide P-C were identical to those reacting with insulin antisera. As the antisera against peptide P-C did not have any cross-reactivities to insulin, glucagon, somatostatin, pancreatic polypeptide, VIP and human C-peptide, the antisera were considered to recognize specifically either peptide P-C related antigen or peptide P-C itself in human pancreatic B-cells. A novel substance, peptide P-C like immunoreactivity, may be present in pancreatic B-cells independent of pro-insulin and insulin.Morphological similarity between the salivary glands and the pancreas has been reported, and amylase, kallikrein and glucagon are present in both. These findings seem to suggest some functional relation between the pancreas and salivary glands. Detection of peptide P-C like immunoreactivity in the pancreas and salivary glands would be a additional support for this idea. Although it is suggested that peptide P-C like immunoreactivity in pancreatic B-cells may play some role in the function of B-cells, its exact pathophysiological role remains obscure.

Abstract. Urinary excretion rate and clearance of α1-acid glycoprotein (orosomucoid), a major serum glycoprotein which is more anionic (pI 2.7) than albumin (pI 4.7) were measured by RIA in timed overnight urine samples from non-insulin-dependent diabetic patients with different urinary albumin excretion rate and from healthy controls. The 50th percentiles of urinary orosomucoid excretion rate in patients with normo-, micro-, and macroalbuminuria were larger than those in healthy controls. Urinary excretion rate and clearance of orosomucoid increased in parallel with increase in albumin excretion rate in diabetic patients with an albumin excretion rate of more than 10 μg/min. On the basis of their levels of urinary orosomucoid excretion, patients with normoalbuminuria of less than 10 μg/min could be divided into two groups, one with a normal and the other with an elevated urinary orosomucoid excretion rate. The findings suggest that kidneys of diabetic patients with an albumin excretion rate of more than 10 μg/min are unable to distinguish the difference in pI between albumin and orosomucoid, and that a subgroup with an elevated orosomucoid excretion rate may be present among diabetics with normoalbuminuria.

Abstract. Salivary peptide P-C like immunoreactivity, originally isolated from human whole saliva has later been found in the human pancreatic B-cells. In the present work an indirect immunofluorescence technique using monoclonal antibodies against isolated salivary peptide P-C was applied to Bouin fixed pancreas and parotid glands to study the possible identity of the two substances. Positive P-C immunofluroescence was found in the serous cells of parotid glands but not in pancreatic B-cells, suggesting that pancreatic P-C substance is not salivary peptide P-C itself, but a substance sharing the common antigenic site with salivary peptide P-C. To examine this, an indirect immunofluorescence technique using polyclonal P-C antisera pre-absorbed with six kinds of synthetic fragments (1–22, 23–44, 23–29, 30–44, 30–38 and 38–44) of salivary peptide P-C was applied to the human pancreas. The result showed that pancreatic P-C substance was a substance which shares the common antigenic site with the 38–44 amino acid residue of salivary peptide P-C. Western blot analysis using extracts of human pancreata further showed that pancreatic P-C substance is not a precursor of insulin but a protein with molecular weight of 11 500 dalton, indicating the presence of a new protein in the insulin secretory granules of human pancreatic B-cells.

Radioimmunoassay using labelled somatostatin-14 revealed that components of somatostatin-28 antisera cross-reactive to somatostatin-14 were removed by absorption of somatostatin-28 antisera with sepharose 4B-somatostatin-14. Indirect immunofluorescence techniques using specific antisera against somatostatin-28 were carried out in normal pancreas, duodenum, a somatostatinoma in the duodenum, and pancreatic tumour cells containing somatostatin-14 positive cells, in order to establish if somatostatin-28 is present in normal and pathological tissues. Somatostatin-28 like immunoreactivity was present in pancreatic islets cells and in the epithelial cells of the duodenum as well as in the duodenal somatostatinoma and in pancreatic tumour cells. Furthermore, cells reacting with specific antisera against somatostatin-28 were identical to those with somatostatin-14 antisera in normal and pathological tissues. The findings suggested that somatostatin-28 like immunoreactivity may be constantly present in the tissues where somatostatin like immunoreactivity was detected using somatostatin-14 antisera. However, further studies are necessary to clarify whether somatostatin-28 and somatostatin-14 were independently present in these tissues, in other words, whether somatostatin-14 may be produced from somatostatin-28 or not, since somatostatin-14 antisera had cross-reactivities to somatostatin-28.

Abstract. In order to clarify the intracellular localization of salivary peptide P-C-like immunoreactivity in human pancreatic B-cells, an immunohistochemical study at electron microscopic levels was carried out by the protein A-gold technique using antisera against insulin and salivary peptide P-C. Both salivary peptide P-C-like immunoreactivity and insulin-like immunoreactivity were present only in the insulin secretory granules of the pancreatic B-cells. However, the former immunoreactivity was lacking in many insulin secretory granules of foetal pancreatic B-cells while the latter immunoreactivity was seen in all insulin secretory granules. Salivary peptide P-C-like immunoreactivity was not found in the other kinds of cells in the islets. In a previous immunohistochemical study at light microscopic level, salivary peptide P-C-like immunoreactivity appeared in a few pancreatic B-cells at about the 16th week of gestation, in an increasing number during gestation, and was seen in all pancreatic B-cells a few months after birth. The present finding together with the above results suggest that absence of salivary peptide P-C-like immunoreactivity in some foetal pancreatic B-cells may be due to the underdevelopment of salivary peptide P-C-like immunoreactivity in each insulin secretory granule. From the examination of cross-reactivity of antisera against salivary peptide P-C to other kinds of salivary peptides and salivary Protein C, and from the results of an indirect immunofluorescence technique using three kinds of antisera including antisera against salivary peptide P-C, salivary peptide P-B and salivary Protein C, it was thought that salivary peptide P-C-like immunoreactivity in human pancreatic B-cells belongs neither to salivary Protein C nor to salivary peptide P-B nor to salivary peptide P-E, but either to salivary peptide P-C itself or to an unknown substance which has common antigenic determinants with salivary peptide P-C, salivary peptide P-B and salivary Protein C. Salivary peptide P-C-like immunoreactivity was not found in the pancreatic B-cells of other mammals. Thus, although a new substance other than insulin is present in the insulin secretory granules of the human pancreatic B-cells, its pathophysiological function remains unclear.

Yamazaki M, Ito S, Usami A, Tani N, Hanyu 0, Nakagawa O. Nakamura H, Shibata A. Urinary excretion rate of ceruloplasmin in non-insulin-dependent diabetic patients with different stages of nephropathy. Eur J Endocrinol 1995;132:681–7. ISSN 0804–4643The level of ceruloplasmin, which is a more negatively charged protein than albumin, was measured by an immunoradiometric assay in timed overnight urine and serum samples from patients with non-insulin-dependent diabetes mellitus and healthy controls. None of the plasma proteins examined showed any cross-reactivity in this assay. A linear correlation was seen between the ceruloplasmin level and the serial dilution of the sample. Western blot analysis using concentrated urine samples showed that the molecular weight of ceruloplasmin in the urine sample was the same as that of ceruloplasmin in the serum and standard samples. These findings indicated that the substance detected by this assay was truly ceruloplasmin. The urinary ceruloplasmin excretion rate (CER) and clearance of ceruloplasmin increased in parallel with the progression of albuminuria. The highest CER was found in macroalbuminuric patients, followed by micro- and normoalbuminuric patients and the healthy control subjects, the differences between the groups being significant. In view of the fact that the isoelectric point of ceruloplasmin (4.4) is more acidic than that of albumin, the present findings suggested that an enhanced CER was due either to the alteration of charge selectivity in the glomerular basement membrane with unaltered tubular function or to a defect of the non-discriminatory pores (shunt pathway) with unaltered tubular function.Seiki Ito, Division of Gerontology, Akita University Hospital, 1-1-1 Hondou, Akita City, Japan 010

By an indirect immunofluorescence technique and silver impregnation, cells reacting with anti-1-18ACTH antiserum were demonstrated in the foetal adrenal medulla only in the 23rd week of gestation. They were not found in the foetal adrenal glands before the 23rd week or after 29th week and after birth. The result was confirmed by immunohistological examination using anti-1-18ACTH rabbit IgG purified by affinity chromatography. No cells in the foetal adrenal medulla in the 23rd week of gestation were stained positively by using anti-β-MSH and anti-β-endorphin antisera. This finding suggested, therefore, that the synthetic mechanism of ACTH-like immunoreacticvity in the foetal adrenal glands was different from that in the pituitary. In view of the findings that some phaeochromocytoma contained ACTH-like immunoreactivity, the present study suggested that the origin of the phaeochromocytoma containing ACTH-like immunoreactivity may be the cells of the foetal adrenal medulla in about the 23rd week of gestation.

Abstract. An indirect immunofluorescence technique using antisera aganist salivary peptide P-C and against salivary Protein C was carried out on the laryngeal, tracheal and bronchial glands to examine whether salivary peptide P-C-like immunoreactivity, recently demonstrated in the serous cells of the human salivary glands, was also present in those of laryngeal, tracheal and bronchial glands and to ascertain whether salivary peptide P-C is a fragment of salivary Protein C or not. Salivary peptide P-C-like immunoreactivity was present in the serous cells of the human laryngeal, tracheal and bronchial glands. Observation of serial sections immunostained with two kinds of antisera revealed that cells reacting with antisera against salivary peptide P-C were identical to those reacting with antisera against salivary Protein C pre-incubated with salivary peptide P-C. The finding implied that salivary peptide P-C and salivary Protein C, originally isolated from human saliva, were also present in the serous cells of tissues other than the salivary glands. Furthermore, analysis of the primary structure of salivary peptide P-C and salivary Protein C together with the present morphological finding suggests that salivary peptide P-C is a COOH-terminal fragment of salivary Protein C. Thus, salivary Protein C and salivary peptide P-C may play some role in the function of the serous cells of the salivary and laryngo-tracheobronchial glands.