Changes in serum lysosomal hydrolases in marasmus.

The activities of several lysosomal hydrolases including beta-glucuronidase, acid phosphatase and hexosaminidase were compared in serum from 19 well-nourished subjects and 13 children (age 5--24 months) who were suffering from marasmus. The marasmic children exhibited growth retardation and muscle wastage but had normal serum protein values and absence of psychomotor retardation or oedema. Significant changes were observed in serum beta-glucuronidase and acid phosphatase activities. Compared to the control group, serum beta-glucuronidase (determined at pH 4.5 using the fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucuronide) was 2.3-fold higher (p less than 0.001) in the marasmic children. In contrast, serum acid phosphatase values were approximately 50% lower (p less than 0.01) in the marasmic population. Serum hexosaminidase values in the two groups under study were not significantly different. Determination of the beta-glucuronidase to acid phosphatase ratio permitted effective discrimination (p less than 0.001) of serum from normal and protein-calorie malnourished children. The finding that the elevated value of the beta-glucuronidase : acid phosphatase ratio (0.64--1.37) decreased to within the normal range of values (0.10--0.43) after nutritional rehabilitation of several marasmic cases indicates that the determination of serum lysosomal hydrolases using fluorogenic substrates might provide a rapid and sensitive quantitative method for objectively evaluating the status of protein-calorie malnourished children and their responsiveness to nutritional therapy.     

Bile lysosomal enzymes: characteristics and pathological significance for various hepatobiliary disorders.

The activities of several glycosidases (beta-galactosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase) were demonstrated in human bile. The enzyme activities are increased about 100 times after exclusion of bile salts and other small molecular compounds by Sephadex G-50 gel filtration. The use of 4-methylumbelliferyl derivatives as substrates was useful as measurement of the bile enzyme activities are not altered in the presence of bile pigments. Enzyme characteristics of bile glycosidases were determined: pH optimum and isoelectric point. The bile glycosidase activities were also measured in various hepatobiliary disorders (cholelithiasis, cancer of gallbladder, acute hepatitis, liver cirrhosis and fatty liver). The glycosidase activities in bile from patients with liver diseases, as well as with cholelithiasis, were generally decreased. Isoelectric focusing patterns of biliary glycosidases were similar for specimens from patients with hepatobiliary disorders as compared to normal.     

Alpha-L-fucosidase and alpha-D-mannosidase activity in the white blood cells in the disease and carrier state of fucosidosis

α-l-Fucosidase and α-d-mannosidase activity in white blood cells from a patient with fucosidosis and her family members were studied.α-l-Fucosidase activity of the patient was completely lacking, whereas the α-l-mannosidase activity was increased and relative thermostability of this enzyme was also observed. Apparent K_m value of α-d-mannosidase was similar in the patient and the control. When the ratio of α-l-fucosidase to α-d-mannosidase activity was calculated, the value of the parents was intermediate between that of the patient and the control.     

Studies on the presence of 25-hydroxyvitamin D in human saliva

Saliva collected from volunteers of both sexes at various times throughout the day was examined for the presence of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The method used was a specific high-performance liquid chromatographic procedure with a minimum detectable limit for 25-hydroxyvitamin D of 0.05 microgram/1, (130 pmol/1 for 25-hydroxyvitamin D3) using 20 ml of saliva. No 25-hydroxyvitamin D could be detected in any of the samples. Examination of larger quantities of saliva (100 ml) by a combination of high-performance liquid chromatography and gas chromatography-mass spectrometry showed the presence of a peak possibly derived from 25-hydroxyvitamin D3, at a concentration not in excess of 65 pmol/l. Using known binding characteristics of 25-hydroxyvitamin D and plasma vitamin D binding protein, and comparing them with similar characteristics for cortisol and plasma cortisol binding globulin and the known salivary concentration of cortisol, simple calculations indicated that a likely concentration of 25-hydroxyvitamin D in saliva would be around 8 pmol/l. Previously reported values ranging from 130-4160 pmol/l (50-1600 micrograms/l) using a simple competitive protein binding assay could not be confirmed.     

Galactose 6-sulfate sulfatase activity in Morquio syndrome

We have prepared a new substrate (o-beta-D-sulfo-galactosyl-(1-4)-beta-D-6-sulfo-2-acetamido-2-deoxyglucosyl- (1-4)-D-[1-3H]galactitol), from shark cartilage keratan sulfate, for the assay of galactose 6-sulfate sulfatase activity. Using this substrate, we found there was a striking deficiency of galactose 6-sulfate sulfatase activity, in addition to the known deficiency of N-acetylgalactosamine 6-sulfate sulfatase, in the cultured skin fibroblasts of patients with Morquio syndrome. Our results could be explained by the hypothesis that accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio syndrome is due to a deficiency of galactose 6-sulfate sulfatase and N-acetylgalactosamine 6-sulfate sulfatase activity, which are necessary for the degradation of these two mucopolysaccharides.     

Phosphatidylcholine content and fatty acid composition of tracheal and gastric liquids from premature and fullterm newborn infants

The phosphatidylcholine content and fatty acid composition of tracheal and gastric liquids from newborn full-term and premature (30–36 weeks) infants were studied. Phosphatidylcholine accounted for about 70% of the total phospholipids in both liquids from the full-term infants but only 55–57% of those from the prematures. There was a significant correlation between tracheal and gastric liquids in the fatty acid composition of phosphatidylcholine. There were significant differences in fatty acid composition between the full-term and premature liquids. These differences were most apparent in the gastric liquid. It is suggested that phospholipid or fatty acid analysis of tracheal or gastric liquids from newborn infants can be used in the assessment of pulmonary maturity.     

Isoelectrofocusing in acetate membrane: the method and application.

Acetate membrane as a new medium for isoelectrofocusing was used in this study. The acetate membrane was specially produced for isoelectrofocusing, and contains a surface active agent. The ampholine solution system and isoelectric condition for this purpose are described. Polymorphic traits as alpha1-antitrypsin in human serum and 6-phosphogluconate dehydrogenase (6-PGD) in human erythrocyte could be identified by this method. The isoelectric patterns and the isoelectric points of these proteins were demonstrated on the acetate membrane.     

Studies on the activities and properties of lysosomal hydrolases in fractionated populations of human peripheral blood cells

Studies have been carried out on the activities and properties of the isozymes of alpha-mannosidase, alpha-glucosidase and beta-glucosidase in granulocytes, monocytes, lymphocytes and platelts from peripheral blood of heatlhy adult donors. The findings reveal the differences in activities as well as a characteristic distribution of the different molecular forms of these lysosomal hydrolases in specific cell types. Therefore, the results obtained with unfractionated total leukocyte smples from different subjects may vary according to the distribution of cell types in the circulation. Granulocytes and monocytes show only the acid alpha-mannosidase activity whereas lymphocytes and platelets show both acid and neutral activities. The specific activity of acid alpha-mannosidase in granulocytes and monocytes is higher than in lymphocytes and platelets. By DEAE-cellulose chromatography, the acid alpha-mannosidase in granulocyte and monocyte extracts elutes as two peaks, but only one peak is seen in lymphocytes. All cell types show both acid and neutral alpha-glucosidase activities. The specific activities of both isozymes are higher in granulocytes and monocytes than in lymphocytes and platelets. Monocytes show a higher acid than neutral activity. All other cell types show a higher neutral activity. Beta-Glucosidase in all cell types is mainly membrane-bound and it can be released by Triton X-100 and sodium taurocholate. Taurocholate also stimulates the beta-glucosidase activity of granulocytes, monocytes and lymphocytes whereas it inhibits the activity of this enzyme in platelets. These results indicate that variations in the total number of leukocytes and in the relative proportion of the various cell types in health and disease may yield inconsistent or unreliable values for enzyme activity in the diagnosis of lysosomal storage disease and in carrier detection.     

Serum cholesterol and bile acid in primary hepatoma.

The close relationship between serum levels of cholesterol and bile acid has been confirmed in 46 patients with primary hepatoma. Serum levels of cholesterol and bile acid are roughly correlated with serum alpha-fetoprotein concentration. Because the relationship between serum cholesterol and bile acid did not exist in common hepatocellular diseases, the results suggest a peculiar sterol metabolism occurring in human hepatoma.     

Serum squalene levels in hepatobiliary diseases

Serum squalene levels did not change in patients with acute hepatitis, chronic active hepatitis and liver cirrhosis, but were significantly reduced in patients with intra- and extrahepatic cholestasis. The ratio of cholesterol to squalene remained normal in patients with acute hepatitis as well as chronic active hepatitis, while being slightly decreased in patients with liver cirrhosis. On the other hand, in patients with cholestasis the ratio was markedly raised. From these observations we confirmed abnormal sterol metabolism in hepatobiliary diseases, and clinical usefulness of the ratio of cholesterol to squalene to distinguish hepatocellular injury and cholestasis.     

A kinetic expression for sodium-lithium countertransport in human red cells

Sodium-lithium counter-transport in human red blood cells may be a potentially useful measurement in studies of hypertension. A kinetic expression describing this counter-transport was derived and evaluated using red cells from nine subjects at various concentrations of intracellular and extracellular Li⁺ and Na⁺. The counter-transport is dependent upon all four concentrations, intracellular Li⁺ and Na⁺ as well as extracellular Li⁺ and Na⁺. We confirm that the maximum Na⁺-Li⁺ counter-transport (V_max) is a property of the individual cells while the half-maximal saturating concentrations (K_{$\text{1}\text{2}$}) for Li⁺ and Na⁺ are the same for all subjects. This expression permits a more thorough understanding of conditions affecting Na⁺ -Li⁺ countertransport measurement.     

Illness behaviour profiles in chronic pain: The auckland experience

Two hundred patients with chronic pain, presenting to the Auckland Hospital Pain Clinic, completed the illness behaviour questionnaire (IBQ) developed by Pilowsky and Spence in Adelaide. These authors have identified 6 taxonomic clusters from a numerical analysis of illness behaviour profiles and have described the characteristics of these groups of patients. This study reports the results of a similar analysis of IBQ scores taken from a larger group of patients and clustered using a variant of the K-means algorithm. Ten clusters were derived. These are described and compared with the groups described by Pilowsky and Spence. Both samples have closely similar illness behaviour profiles. Comparisons of the clusters reveal more similarities than differences and we conclude that this independent replication of the Pilowsky and Spence study enhances the validity of the IBQ and the clusters described.     

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.     

Partial deficiency of acid lipase with storage of triglycerides and cholesterol esters in liver. Genetic variant of wolman's disease?

Triglycerides and cholesterol esters accumulated in the liver of a patient showing failure to thrive, hepatomegaly and anemia. Acid lipase activity was low but not completely deficient in liver and leukocytes. Correlations of clinical and biochemical data are discussed with special reference to Wolman's disease and cholesterol ester storage disease.     

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.     

Evolution of serum copper and zinc levels in burn patients (author's transl)]

The study of copper and zinc in 35 patients, over periods of up to 21 days following thermal injury, is marked by an important and early fall in serum copper and zinc levels. The maximum intensity of the biological disturbance appears about two days after the injury. Reversion to near normal values coincides with recovery from the large part of the lesions. The comparison of the evolution of serum levels does not permit any definite relationship to be established between copper and zinc metabolism.     

Radioimmunoassay of serum group I and group II pepsinogens in normal controls and patients with various disorders

A radioimmunoassay (RIA) for human group I pepsinogens (PgI) in serum was developed, using Pgl purified from gastric mucosa. The sensitivity (0.7 μg/1) and reproducibility of the assay were satisfactory for clinical use. In normal controls, total serum pepsinogen (T-Pg) level was 58.9 ± 31.7 μg/1 (mean ± SD) (Pgl, 43.6 ± 25.0 μg/1; Pgll, 15.3 ± 11.1 μg/1). Peptic ulcer cases had elevated T-Pg levels (gastric ulcer, gastroduodenal ulcer and duodenal ulcer, in increasing order of magnitude). T-Pg levels were not useful for diagnosis of peptic ulcer because of a large overlap with normal controls. T-Pg levels were low in patients with gastric polyp and in aged subjects. In these groups, the decrease of Pgl was more marked than that of PgII.     

Measurement of unconjugated testosterone, 5alpha-dihydrotestosterone and oestradiol in human urine

Radioimmunoassay methods for measuring unconjugated testosterone, 5α-dihydro testosterone and oestradiol in human urine are described. The methods have been evaluated and precision, accuracy, recovery and specificity found to be satisfactory. Normal males had mean (range) 24-h values of 377 (76–1154) ng, 138 (51–408) ng and 8.9 (3.2–19) ng for testosterone, 5α-dihydrotestosterone and oestradiol, respectively. Normal females in the early follicular phase of the menstrual cycle had mean (range) 24-h values of 92 (33–260) ng, 39 (11–82) ng and 24 (5.7–75) ng for testosterone, 5α-dihydro testosterone and oestradiol, respectively.Urinary levels of the unconjugated hormones in normal females on oral contraceptives, a small group of hirsute females, and a few normal postmenopausal women are given. While the postmenopausal women had similar values for the androgens to the normal female group the hirsute group had much higher average values which were widely scattered and overlapped with the normal group. The women on oral contraceptives had higher excretion of both androgens than the normal group. There was no difference between values in night and day collections. A highly significant overall correlation (p < 0.001) was found between testosterone and 5α-dihydro testosterone values. Both androgens showed a significant correlation (p < 0.02) with urinary creatinine excretion. Unconjugated oestradiol levels showed a poor correlation with the levels of either androgen or the creatinine excretion. Simultaneously obtained serum levels within each group correlated poorly with the urinary levels.     

Enzymic determination of urinary free l-fucose

Conditions necessary for the precise measurement of free l-fucose in urine by an enzymic method using l-fucose dehydrogenase have been studied. The normal urinary levels of l-fucose were 16.4 ± 9.1 μg/ml in children (22.2 ±6.5 μg/mg of creatinine) and 17.7 ± 8.5 μg/ml in adults (16.6 ± 5.7 μg/mg of creatinine). There was a close correlation between the concentration of free l-fucose and that of creatinine. A close correlation was also found between the concentration of free l-fucose and α-l-fucosidase activity. Thus, it was suggested that the free l-fucose in urine reflected the metabolism of l-fucose or l-fucose-containing glycoconjugates.In a preliminary screening test, several urine samples which showed high concentrations of free l-fucose, namely cases of “fucosuria” were found.