The permeability of the blood-brain barrier to protein in the post-hypoxic cerebral edema of the rat

The permeability of the blood-brain barrier to protein was studied in the post-hypoxic cerebral edema of rats, using horseradish peroxidase (HRP) as a tracer protein. Under halothane anesthesia, HRP was injected intravenously. Then the animals were exposed to hypoxic-ischemia by inhalation of 5% oxygen in nitrogen and temporary occlusion of the left common carotid artery for 10-30 min. Twenty-four, 48 or 72 h after the hypoxic experiments, the animals were anesthetized with sodium pentobarbital and their body tissues were fixed by perfusion with a mixture of glutaraldehyde and p-formaldehyde before the autopsy. No brain edema was observed in the animals exposed to hypoxia for 10 min, but the animals exposed to hypoxia for 20-30 min revealed a left hemispheric brain edema. The brain was immediately immersed in the same fixatives, and then sectioned with a cryostat. The sections were stained histochemically according to the method of Graham and Karnovsky (1966) to identify the distribution of the HRP. HRP was not found even in the edematous hemisphere. In this type of brain edema, it is suggested that there was no acceleration of permeability of blood-brain barrier to serum proteins whose molecular weights were larger than HRP, and therefore it would have no causative relationship with the production of brain edema.     

Low urinary excretion of heparan sulfate in three patients with Lowe's syndrome

Urinary glycosaminoglycans were isolated with the cetylpyridinium chloride (CPC) precipitation method and the excretion of individual species of urinary glycosaminoglycans in three patients with Lowe's syndrome was compared with that of age-matched control children by means of electrophoresis on cellulose acetate membranes and by quantification of hexosamine contents. Total daily excretion of urinary glycosaminoglycans in the patients seemed to be normal, but the relative excretion of urinary heparan sulfate was significantly reduced and ranged from 26 to 46% of the age-matched control mean, when calculated on the basis of relative glucosamine content in urinary glycosaminoglycans. Although electrophoretograms of urines from patients with Lowe's syndrome suggested some excess of low sulfated chondroitin sulfate corresponding in mobility to dermatan sulfate, the enzymatic subunit assay employing chondroitinases did not disclose any significant differences in the excretion pattern or in the degree of sulfation of chondroitin sulfate isomers between Lowe's syndrome and control children.     

Serum levels of thyroid hormones in liver diseases

Concentrations of thyroid hormones and thyrotropin (TSH) were measured in sera of clinically euthyroid patients with various liver diseases and compared with normal controls. The mean serum concentration of 3,3',5'-triiodothyronine (reverse T₃, rT₃) was significantly increased in patients with decompensated liver cirrhosis (p < 0.01). This increase seemed to be dependent upon the hepatic damage, although it was not significant in patients with acute hepatitis, chronic hepatitis and compensated liver cirrhosis. The mean serum concentration of 3,3',5-triiodothyronine (T₃) was significantly decreased in patients with decompensated liver cirrhosis (p < 0.05). However, in patients with acute hepatitis, chronic hepatitis and compensated liver cirrhosis, the mean concentration of T₃ was above the normal. The mean value of rT₃/T₃ ratios in patients with acute hepatitis, chronic hepatitis and compensated liver cirrhosis were similar to that of normal controls, but in patients with decompensated liver cirrhosis, the mean value of rT₃/T₃ ratios was markedly higher than that of normal controls. The rT₃/T₃ ratios have little or no correlation with some standard liver function tests.These results suggest that marked alterations of peripheral conversion of thyroxine (T₄) to rT₃ or T₃ may be found only in a state of decompensated liver cirrhosis among the various liver diseases.     

Plasma 1,25(OH)2 vitamin d measured by radioimmunoassay and cytosol radioreceptor assay in normal subjects and patients with primary hyperparathyroidism and renal failure

Antibodies to 1α,25-dihydroxy vitamin D₃ 25-hemisuccinate linked to albumin were produced and an immunoassay for 1,25-dihydroxy vitamin D developed. Plasma 1,25-dihydroxy vitamin D concentrations were compared using an immunoassay and cytosol radioreceptor assay. Both assays gave comparable results but the immunoassay was more reproducible, slightly more sensitive and had a lower detection limit.Using the immunoassay the plasma 1,25-dihydroxy vitamin D was 110.5 pmol/l (S.D. 29.4) in normal subjects; there was no difference between males and premenopausal females. It was negatively related to plasma phosphate. In renal failure and primary hyperparathyroidism plasma 1,25(OH)₂D was positively related to radiocalcium absorption. Following 1 and 2 μg of 1,25-dihydroxy vitamin D₃ given orally the peak plasma concentration occurs within 12 h.     

Prolyl hydroxylase activity in human normal skins and post-burn scars.

Post-burn granulation tissues showed an extraordinary high prolyl hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase) activity, 25 to 50 times higher than the mean value of human normal skins from the frontal thighs of 15 subjects, 385 +/- 247 (S.D.) cpm/min/g of wet weight tissue. The activity in the scars decreased sharply within 4 to 5 months, and then gradually decreased to the normal range after 2 years or so. Well-aged scars tended to show lower values than the mean value of normal skins.     

Effect of dithiothreitol on the reaction of renin and angiotensinogen

Effects of several sulfhydryl reagents on the reaction of renin and angiotensinogen were investigated by measuring the production of angiotensin I. Renin and angiotensinogen were prepared from the kidneys of dogs and from plasma of nephrectomized dogs, respectively. The amount of angiotensin I generated was increased with application of dithiothreitol, dithioerythritol and 2,3-mercapto-1-propanol. The greatest enhancement of the generation of angiotensin I was observed with dithiothreitol. Dithiothreitol had no influence on renin activity, on radioimmunoassay for angiotensin I and the recovery of synthetic angiotensin I from the incubation medium. When heterologous angiotensinogen was used, a great enhancement of generation of angiotensin I was observed. Dithiothreitol did not accelerate the reaction following replacement of the natural substrate, angiotensinogen, with the synthetic tetradecapeptide as substrate. It is postulated that dithiothreitol augments angiotensin I generation by acting on the angiotensinogen and not on the renin.     

Action of human pancreas alanine aminopeptidase on biologically active peptides: kinin converting activity

A group of 15 biologically active peptides were studied with respect to their susceptibility to chain shortening by human pancreas alanine aminopeptidase. Those susceptible were somatostatin, melanocyte stimulating hormone, fibrinopeptide A, eosinophilotactictetrapeptide, lysyl-bradykinin, and methionyl-lysyl-bradykinin. The latter two were selected for further study. Direct identification and determination of the reaction products, lysine and/or methionine, were undertaken to establish unequivocally the kinin-converting activity of human pancreas alanine aminopeptidase, which exhibited a pH optimum at pH 7.9. The Km and kcat values for this enzyme for lysyl-bradykinin were 57 mumol/l and 3000 min-1, respectively. The corresponding values for this enzyme for methionyl-lysyl-bradykinin were calculated to be 49 mumol/l and 16000 min-1, respectively. Bradykinin itself is extremely resistant to hydrolysis by this pancreatic enzyme.     

Neuraminidase in mucolipidoses: normal activity in frozen autopsy tissues from three patients with I-cell disease and adult beta-galactosidase deficiency.

Neuraminidase was assayed in the frozen autopsy tissues from three patients with I-cell disease and an adult patient with cherry-red spots, myoclonus, cerebellar ataxia and beta-galactosidase deficiency. Both diseases showed normal neuraminidase activity toward neuramine lactose and fetuin in cerebral gray matter, liver and kidney. These results suggest that the neuraminidase deficiency is limited only to some tissues and that this biochemical abnormality is not caused by a primary genetic mutation in these diseases.     

Purification and properties of urinary alkaline ribonucleases from patients with nephrotic syndrome.

Four urinary alkaline ribonucleases (RNase, EC 3.1.4.22) were purified from patients with nephrotic syndrome using phosphocellulose, DEAE-cellulose and Sephadex G-75 chromatographiy. These enzymes were designated as RNases 1--4, respectively, in order of elution on phosphocellulose chromatography. The respective purification of each fraction was 41-, 23-, 34- and 27-fold with a total recovery of 25%. The pH optima of these RNases were around 8.5 with Tris/HCl buffer and the reaction was activated by mono- and divalent cations, such as Na+, K+, Mg2+ and Ca2+, but inhibited by Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of reaction. The molecular weights of RNases 1--4 were estimated by gel filtration as 45 000, 32 000, 20 000, and 13 000, respectively. Each enzyme hydrolyzed pyrimidine nucleotides preferentially with higher affinity for poly(C) than poly (U) as determined with synthetic polymers and was free from other nucleolytic enzymes. The patients with renal disorders excreted one to four RNases in urine and the number of enzymes increased as the concentration of urinary protein increased. On the other hand, normal subjects excreted a single fraction essentially identical to RNase 1.     

Determination of sorbitol in erythrocytes of diabetic and healthy subjects by capillary gas chromatography

A gas-liquid chromatographic method, using a fused silica capillary column, for the determination of red cell sorbitol is described. The capillary column gives complete resolution of the polyols xylitol, inositol, mannitol, sorbitol and galactitol, even when the glucose peak in the red cell chromatogram is dominating. The identity of sorbitol, and its single elution from the capillary column has been confirmed by mass spectrometry. Recovery of sorbitol from various red cell samples was 101% +/- 3.2 (mean +/- SD, n = 7). Precision, estimated from duplicate diabetic red cell sorbitol analyses was CVdup = 3.5% (n = 18) and from run to run analyses CVinterassay = 4.0% (n = 6). Sorbitol levels determined in erythrocytes of 19 healthy subjects were 5.9 +/- 1.6 nmol/ml red cells and in erythrocytes of 18 insulin-dependent diabetics 17.8 +/- 8.2 nmol/ml red cells (means +/- SD). The method described offers a reliable and specific tool to study in vivo polyol pathway activity in relation to some diabetes-associated complications.     

Improved determination of serum calcium with 2-cresolphthalein complexone

This paper describes experimental conditions for reproducible measurement of sodium-lithium countertransport in red blood cells. The assay is sensitive to temperature (10% per °C at 37°C) and the condition of the red cells; it is affected little by changes in intracellular lithium over the range 2–7 mmol/1 or by red cell concentrations with laboratory hematocrits of 0.03 to 0.07. Repeated measurements of the same subjects give day-to-day coefficients of variation of 10% or less. The mean difference for interlaboratory comparisons is 11%.     

Lysyl oxidase activity in human normal skins and postburn scars.

Lysyl oxidase activity of human normal skins derived from the frontal thighs of 33 subjects showed large variations and the mean value was 11 455 +/- 7 172 (S.D.) cpm/g of wet weight tissue. The age of lesion affected the lysyl oxidase activity in postburn scars. Granulation tissues showed a fairly low activity; however, the activity increased sharply within 2--3 months, and reached a significantly higher value than that of normal skin. The high level of activity continued for up to 2--3 years, then gradually decreased to normal range after 5 years or so. Lysyl oxidase activity was detected only after 4 M urea treatment of tissues. Benzylamine oxidase activity also showed large variations in both normal skins and postburn scars, with mean values of: 0.128 +/- 0.077 (S.D.) and 0.145 +/- 0.090 (S.D.) mmol/g of wet weight/h, respectively. No correlation was observed between lysyl oxidase and benzylamine oxidase activities. The granulation tissues showed significantly high values of benzylamine oxidase activity in contrast to the low values of lysyl oxidase activity.     

Study of serum beta-2 microglobulin levels in breast cancer patients.

Serum beta-2 microglobulin levels were measured in normal individuals and in breast cancer patients. It was observed that there was a significant rise in levels especially in advanced stages of the disease. It was concluded that patients with a beta-2 microglobulin/creatinine ratio of greater than 3.8 were likely to have metastatic breast cancer.     

Erythrocyte spectrin in Duchenne muscular dystrophy

The primary structure of erythrocyte spectrin bands I and II from controls and patients with Duchenne muscular dystrophy was compared by 2-dimensional peptide mapping. ¹²⁵I-labelling was done either by the chloramine-T method or using the Bolton and Hunter reagent followed by treatment with trypsin or chymotrypsin, resulting in four different peptide maps from each band of spectrin. Although all the peptide maps of band I were considerably different to those of band II, there were no consistent differences in the maps of bands I and II from controls compared to the corresponding maps from patients with Duchenne muscular dystrophy.     

The use of tears for diagnosis of GM1 gangliosidosis.

The properties of beta-galactosidase of tears were investigated and the standard assay system was accomplished. The pH optimum was 4.2. The enzyme had a KM of 8.3 X 10(-4) M. The activity was stimulated by chloride ions and slightly stabilized by bovine serum albumin. The activities of normal individuals were 205 +/- 80 (S.D.) nmol/h/ml. The activity in the tears of the patient with GM1 gangliosidosis decreased to about 20% of normal control and this disease could be diagnosed by the assay of beta-galactosidase in tears.     

A gas chromatography-mass spectrometry assay of human plasma catecholamines

Dopamine, norepinephrine and epinephrine have been determined by mass fragmentography in plasma of normo- and hypertensive patients. New derivatives of norepinephrine and epinephrine have been prepared, allowing the simultaneous reading of the base peaks of the three amines and of their internal standards with a magnetic-sector mass spectrometer.     

The presence of creatine kinase bb isoenzyme in patients with prostatic cancer

Creatine kinase BB isoenzyme (CK-BB) was detected in abnormal amounts in serum samples from 11 of 46 patients with Stage D carcinoma of the prostate by electrophoresis. Thirteen of 46 Stage D patients had elevated acid phosphatase values and 10 of these 13 had elevated CK-BB. CK-BB elevations were less frequent in earlier stages of prostatic cancer; Stage C: 0 of 35, Stage B: 1 of 26, Stage A: 0 of 3 and none in a group of 35 with BPH, prostatitis and bladder cancer. Results of CK-BB by a specific radioimmunoassay correlated well with those obtained by electrophoresis in most cases. Several patients were followed over time and data on CK-BB is presented for this interval. The origin of the CK-BB is still unclear. The BB isoenzyme predominates in prostatic tissue and CK-BB is the fetal form of the enzyme in human muscle and myocardium. The increase in serum CK-BB may be related to increased release of the isoenzyme, either from the prostate itself or from a metastatic lesion, or may represent a release of the fetal form of the enzyme from dedifferentiated tumor tissue.     

Respiratory mucous secretions in patients with cystic fibrosis: relationship between levels of highly sulfated mucin component and severity of the disease

The tracheobronchial secretions from cystic fibrosis patients contained higher levels of protein, DNA and sialic acid than the tracheobronchial secretions from healthy donors. In contrast, the neutral hexose content in CF secretions was strikingly lower than in secretions from normal subjects. The levels of neutral hexose and sialic acid in the CF secretions were found to increase with increasing severity of the disease. The alterations in the levels of these chemical parameters in the secretions of patients with increased disease severity are as a result of increased levels of the mucin content of the secretions, especially of the highly sulfated mucin component. Since mucins are considered, to a large extent, responsible for the viscoelastic properties of the secretions, the enhanced levels of the highly sulfated mucin component in the secretions of the patients with increased disease severity, may contribute to altered rheological properties and hence decreased mucociliary transport of the secretions.     

Ectopic production of a salivary type amylase by adenocarcinoma cells: Demonstration by a culture technique

Characterization of an elevated amylase activity in ascitic fluid obtained from a patient with carcinomatous peritonitis is described; ninety-one per cent of the increased amylase activity in the fluid was of salivary type and the remainder of pancreatic type, when studied by ion-exchange chromatography. Culture of ascitic cells successfully demonstrated morphologically characterized tumor cells surviving for at least 31 days. During that period, significant amylase activities were detected in the culture media, and the isozyme pattern was a single band whose electrophoretic mobility corresponded to salivary amylase.The data obtained indicate that the ascites amylase of salivary type was produced ectopically by the tumor cells.     

A fluorescent artefact resembling BB-creatine kinase in sera of patients with prostatic disease.

A fluorescent zone with mobility towards the anode almost equal to that of human BB-creatine kinase has been detected after electrophoresis on cellulose acetate of sera from each of 28 patients with prostatic carcinoma. The zone is not due to the BB isoenzyme and its appearance does not depend on the presence of substrates of creatine kinase. It therefore appears to be a further example of a fluorescent artefact resembling a creatine kinase ieoenzyme. These observations indicate a need for caution in assessing the possible value of BB-creatine kinase in patients with prostatic disease.