Diagnosis of Tay-Sachs disease using [3H]N-acetylneuraminic acid labelled GM2 ganglioside as substrate

G_M2 ganglioside labelled with tritium in the N-acetylneuraminic acid moiety was prepared and used to measure β-hexosaminidase A activity in cultured human skin fibroblast extracts. The latter convert this substrate to the correspondingly labelled G_M3 ganglioside which can easily be separated from the substrate by thin-layer chromatography. No cleavage of the N-acetylneuraminic acid group was observed under our conditions.

Two methods are described for the determination of G_M2-β-hexosaminidase A activity in fibroblasts. The application of these methods to the diagnosis of Tay-Sachs disease is discussed.     

LYSOSOMES OF THE ARTERIAL WALL : I. ISOLATION AND SUBCELLULAR FRACTIONATION OF CELLS FROM NORMAL RABBIT AORTA

Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks'' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-β-glucosaminidase, N-acetyl-β-galactosaminidase, β-galactosidase, β-glucuronidase, α-mannosidase, β-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor. Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-β-glucosaminidase and β-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5''-nucleotidase and leucyl-β-naphthylamidase (plasma membrane), and catalase (? peroxisome). This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological.     

Absence of increased urinary excretion of adenosine-deaminase-binding protein by patients with chronic renal tubular malfunction

The urinary excretion of adenosine-deaminase-binding protein, a constituent of the brush border of proximal renal tubule cells, has been investigated in 39 patients with disorders associated with malfunction of the renal tubules, and its excretion has been compared with that of two low molecular mass plasma proteins and an enzyme derived from renal tubular cells.

None of the 36 patients with disorders associated with chronic renal tubular malfunction were found to be excreting significantly increased quantities of adenosine-deaminase-binding protein but 30 had increased excretion of retinol-binding protein, ₁-microglobulin, or N-acetyl-β-D-glucosaminidase.

Measurement of urinary adenosine-deaminase-binding protein may be useful in the assessment of acute renal tubular injuries but it is not of value in the detection of chronic renal tubular disorders.     

Identification of β-aminoisobutyric acid in uremic serum

An unidentified ninhydrin-positive substance found in uremic sera but not found in normal sera was isolated by gel-filtration through Sephadex G-75 followed by high voltage paper electrophoresis (pH 3.5), and identified as β-aminoisobutyric acid using paper chromatography and automated amino acid analyzer.

The quantitative determination of β-aminoisobutyric acid in serum revealed that the level of β-aminoisobutyric acid in uremic sera was much higher than that of normal sera.

Gas chromatographic determination of the enantiomorphs of β-aminoisobutyric acid showed that uremic sera contain R- and S-isomers of the amino acid, but with the R-isomer as the dominating form.     

(Mg2+ + Ca2+)-ATPase activity in plasma membrane enriched preparations of human skin fibroblasts: decreased activity in fibroblasts derived from cystic fibrosis patients

Plasma membrane enriched preparations obtained from cultured human skin fibroblasts by differential centrifugation and sucrose density centrifugation techniques were found to contain a Mg²⁺-dependent Ca²⁺-stimulated ATPase activity. The specific activity of the (Mg²⁺ + Ca²⁺-ATPase present was 4–5-fold higher than that present in crude membrane preparations and 80–100-fold higher than that present in homogenates. The (Mg²⁺ + Ca²⁺-ATPase activity of both crude and plasma membrane enriched preparations of cultured fibroblasts from cystic fibrosis patients was significantly reduced compared to that activity observed in age-matched controls. This study corroborates our previous observations made in crude homogenate preparations of fibroblasts and indicates another cell type where (Mg²⁺ + Ca²⁺-ATPase activity may be altered in cystic fibrosis.     

β-Glucuronidase-resistant bilirubin glucuronide isomers in cholestatic liver disease — Determination of bilirubin metabolites in serum by means of high-pressure liquid chromatography

“Direct reacting bilirubin” in serum of patients with cholestatic liver disease and in serum of bile duct-ligated rats consists of a complex mixture of bilirubin metabolites. These metabolites were studied by means of high-pressure liquid chromatography.

Bilirubin glucuronides in normal bile are β-glycosidic 1-O-acyl conjugates which are completely hydrolyzed on incubation with β-glucuronidase. Cholestatic serum contains glucuronide and non-glucuronide bilirubin metabolites. The glucuronides were only partially hydrolyzable with β-glucuronidase. Compernolle et al. [11] showed that the 1-O-acyl bond of bilirubin glucuronides is labile and prone to migrate from the C₁ position at the glucuronosyl residue to positions C₂, C₃ and C₄. The isomerisation products are non-β-glycosidic, β-glucuronidase-resistant conjugates. The main β-glucuronidase-resistant conjugates in cholestatic serum were characterized as: non-β-glycosidic bilirubin monoglucuronide, non-β-glycosidic diglucuronide and a diglucuronide isomer with β-glycosidic and non-β-glycosidic glucuronosyl groups. Moreover, a substantial amount of bilirubin monoglucoside monoglucuronide was detected in cholestatic human serum.     

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl-β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl-β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.     

Application of epithelial cells in culture to the biochemical studies of lysosomal hydrolase deficiencies

Epithelial cells are readily obtained when the primary culture of skin fibroblasts is established. The major lysosomal hydrolase activities in normal epithelial cells are lower than those in normal fibroblasts. The primary deficiency of lysosomal enzymes can be detected both in epithelial cells and in cultured skin fibroblasts. However, epithelial cells of I-cell disease show only β-galactosidase deficiency. This result indicates that epithelial cells have different biochemical properties from skin fibroblasts and they may be similar to those of visceral organs. Therefore, cultured epithelial cells seem to be useful for the studies on lysosomal hydrolase deficiencies.     

Further studies on thermosoluble proteins in serum and urine

Thermosoluble proteins have been isolated from normal human serum and urine. The following proteins were identified in both media : tryptophan-rich prealbumin, ₁-acid glycoprotein, ₁-antitrypsin, ₂HS-glycoprotein, Zn-₂-glycoprotein, transferrin, hemopexin, β₂-glycoprotein I, β₂-glycoprotein III, γG-fragments. Thermosoluble proteins in serum are exclusively related to the above components, while in urine 18% of heat-resistant proteins belong to other tissue proteins. Quantitative determination has shown that ₁-acid glycoprotein and tryptophan-rich prealbumin constitute the major part of thermosoluble proteins in serum, while ₁-acid glycoprotein and Zn-₂-glycoprotein are the major components in urine.     

Contrast medium causes the apparent increase in β-endorphin levels in human cerebrospinal fluid following brain stimulation

Levels of β-endorphin immunoreactivity in cerebrospinal fluid were measured in 12 chronic pain patients undergoing the surgical implantation of an electrode into the periventricular gray matter. Cerebrospinal fluid fractions were collected following placement of a cannula into the third ventricle, following injection of metrizamide contrast medium into the ventricles, following implantation of the electrode, and following electrical stimulation. A second set of samples was collected on a non-surgical day before and after stimulation.

Levels of β-endorphin immunoreactivity increased significantly from baseline levels to post-electrode implantation in one group of patients, but no significant change was seen following the onset of stimulation. Immunoreactivity increased significantly following metrizamide injection in a second group and was still elevated, in comparison to baseline, following electrode placement, but no increase was seen following the onset of stimulation. Levels of immunoreactive β-endorphin did not increase in either group after stimulation on a post-surgical day, despite consistent reports of pain relief. Addition of metrizamide or a related contrast medium, iothalamate meglumine (Conray) to the β-endorphin radioimmunoassay revealed that both compounds interfered with antigen-antibody binding and also quenched the gamma radiation emitted by iodinated peptide ligands. Due to these combined effects, the contrast media alone produced results similar to those of the β-endorphin standard. Moreover, similar observations were made when contrast media were incorporated into radioimmunoassays for met-enkephalin, dynorphin and cholecystokinin octapeptide.

These findings indicate that increased levels of β-endorphin in cerebrospinal fluid are not directly associated with patient report of pain relief following periventricular gray stimulation. This study also suggests that the previously reported association between periventricular gray stimulation and elevated levels of β-endorphin immunoreactivity in ventricular cerebrospinal fluid are artifactual due to the interaction of contrast media with the radioimmunoassay.     

Application of a GM1 ganglioside β-galactosidase microassay method to diagnosis of GM1 gangliosidosis

The enzymatic diagnosis of G_M1 gangliosidosis, including the diagnosis of heterozygosity, requires a microassay of G_M1 ganglioside β-galactosidase activity in lymphocytes and cultured skin fibroblasts. We have adopted high-performance liquid chromatography (HPLC) to the assay of this enzyme and can measure the activity in crude samples fluorometrically. Reaction conditions were examined to determine those optimal for the assay of G_M1 ganglioside β-galactosidase activity in lymphocyte and skin fibroblast homogenates. Under these optimal conditions, reduced enzymatic activities could be detected in lymphocytes and cultured skin fibroblasts from three patients with G_M1 gangliosidosis. Thus, this assay can be used for the diagnosis, rather than the usual assays employing radioactive or artificial substrates.     

The effect of mild therapeutic hypothermia on renal function after cardiopulmonary resuscitation in men

Background: Mild therapeutic hypothermia (MTH) improves neurological outcome in patients after cardiac arrest. From animal and human studies it appears that hypothermia impairs renal function. The aim of this study was to examine the effects of MTH on renal function in humans. Methods: Patients were participants recruited in one of the centres of the hypothermia after cardiac arrest-multicenter trial. We measured serum creatinine and creatinine clearance (C_Cr) within 24 h of MTH, at 4 hourly intervals. Patients were followed for acute renal failure and need for renal supportive therapy for 28 days. Results: We included 60 patients (32 hypothermic, 28 normothermic). Median serum creatinine on admission was [{119 μmol/l (IQR 108–133)} {1.35 mg/dl (IQR 1.22–1.50)}] in hypothermic and [{114 μmol/l (IQR 99–131)} {1.29 mg/dl (IQR 1.12–1.48)}] in normothermic patients, and decreased to [{69 μmol/l (IQR 62–84)} {0.78 mg/dl (IQR 0.70–0.95)}] in the hypothermic group and to [{88 μmol/l (IQR 71–123)} {1.00 mg/dl (IQR 0.80–1.39)}] in the normothermic group within 24 h. C_Cr was decreased on admission. Within 24 h C_Cr improved to normal values in normothermic patients [1.53 ml/s (IQR 1.15–2.35) {92 ml/min (IQR 69–141)}] and remained low in hypothermic patients [0.88 ml/s (IQR 0.63–1.38) {53 ml/min (IQR 38–83)}] (P=0.0006). No difference was found between the groups in the development of acute renal failure or the need for renal supportive therapy. Conclusion: Twenty four hours of MTH was associated with a delayed improvement in renal function. This was not reflected in the serum creatinine values, which were low in the hypothermic group. This transient impaired renal function appeared to be completely reversible within 4 weeks.     

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3, 17-dihydroxy-5β-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3, 11β-dihydroxy-5-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P₃) and 11-keto-pregnanetriol (11-keto-P₃). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3ξ, 16ξ, 17ξ, 20ξ-pregnanetetrol (16-OH-P₃) and surprisingly also a 21-hydroxylated compound, namely 3β, 20, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency.

The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of .the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.     

Immunochemiluminometric assays (ICMA) specific for growth hormone releasing hormone 1-44 NH2 and 1-40 OH.

We describe specific two-site immunochemiluminometric assays able to directly measure human growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH concentrations in unextracted plasma. A common N-terminal antibody was purified from polyclonal rabbit antisera to growth hormone-releasing hormone 1–44 NH₂ on a growth hormone-releasing hormone 1–29 NH₂ linked affinity column and labelled with chemiluminescent acridinium ester. C-terminal specific monoclonal antibodies to growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH were raised in Balb/C mice and used as solid phase antibodies. Assay of fasting specimens from normal individuals gave medians (and ranges) of 23 pg/ml (2–200) and 30 pg/ml (3–134) for growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH, respectively. Samples from a series of acromegalics showed that most have values in the normal range though median values were higher, 56 pg/ml for growth hormonereleasing hormone 1–44 NH₂ (P < 0.001) and 52 pg/ml for 1–40 OH (P < 0.001). Using these assays it will be possible for the first time to directly study the physiology and pathophysiology of these two peptides.     

Urinary albumin determination by the immediate bromcresol green method

It has been demonstrated recently that the reaction of serum samples with bromcresol green (BCG) reagent proceeds in two steps. Albumin is responsible for the immediate reaction while other serum proteins produce the slow reaction.

In this paper the immediate BCG reaction has been used for the determination of urinary albumin concentration in patients with proteinuria by a slightly modified method with a primary pH adjustment of the urine and the use of a urine blank.

Comparison of the immediate BCG method (γ) with Laurell “rocket” technique (χ) gave the following equation: γ = 17.2 +1.006χ (n = 98; r = 0.99) mg/1. The coefficient of variation (within-day), C.V. (%), ranged between 0.9 and 2.7% depending on the albumin concentration. It is thus possible to carry out rapid, accurate and precise albumin determinations in urine samples using this simple method.     

Purification and serological studies of human alpha-L-fucosidase in the normal and fucosidosis states.

An antiserum has been raised to purified alpha-L-fucosidase. Levels of cross-reaction with serum of two unrelated fucosidosis patients and normal individuals with low activity are consistent with the presence of very low amounts of normal enzyme. Similarly no cross-reacting material could be found in cultured fibroblasts from fucosidosis patients. It is deduced that in these cases there is no production of mutant enzyme in quantities comparable to normal levels. Some observations on the interrelations of fucosidases I and II are reported.     

Effects of N-acetylcysteine in endotoxic shock

: The release of oxygen-free radicals has been implicated in both peripheral vascular and myocardial alterations of septic shock. N-Acetylcysteine (N-AC), a substrate for the production of glutathione, has potent antioxidant effects. As a nitrosothiol, it may also improve capillary blood flow. We studied the effects of N-AC in a dog model of endotoxic shock.

: Ten pentobarbital-anesthetized, mechanically ventilated dogs were randomly assigned to receive either N-AC (150 mg/ kg loading dose in 1 hour, followed by 20 mg/kg · h maintenance dose) or D5W. After the loading dose, each dog received 3 mg/kg Escherichia coli endotoxin intravenously. After 30 minutes, saline infusion was started to restore and maintain baseline filling pressures.

: The loading dose of N-AC increased Do₂ significantly (from 661 ± 54 to 914 ± 190 mL/min, P < .05), but Vo₂ remained stable. After the administration of endotoxin, fluid challenge restored cardiac output to baseline, in both groups. Hemoglobin and, thus, Do₂ were slightly lower in the N-AC-treated dogs, but Vo₂ was similar in both groups. At the end of the study, O₂ER was significantly higher in the N-AC-treated dogs than in the control dogs. Blood lactate levels fell more rapidly in the N-AC dogs than in the control dogs. Blood lactate levels returned to normal in the N-AC dogs but not in the control dogs. Tumor necrosis factor (TNF) also decreased significantly in the N-AC dogs but remained elevated in the control dogs.

: These data indicate that N-AC administration in endotoxic shock is well tolerated, may increase oxygen availability to the tissues, and is associated with an attenuation of TNF release.     

Impaired degradation of keratan sulfate in GM1-gangliosidosis

We have prepared a new radiolabeled substrate (galactose-N-acetylglucosamine 6-sulfate-[1-³H]galactitol), from shark cartilage keratan sulfate, for an assay of acid β-galactosidase activity. Using this substrate, we found that there was a striking deficiency of β-galactosidase activity in the cultured skin fibroblasts of patients with GM₁-gangliosidosis. However, there seemed to be no quantitative differences in residual enzyme activity between type 1 and type 2 GM₁-gangliosidosis.     

The Pharmacological Chaperone N-butyldeoxynojirimycin Enhances Enzyme Replacement Therapy in Pompe Disease Fibroblasts

In spite of the progress in the treatment of lysosomal storage diseases (LSDs), in some of these disorders the available therapies show limited efficacy and a need exists to identify novel therapeutic strategies. We studied the combination of enzyme replacement and enzyme enhancement by pharmacological chaperones in Pompe disease (PD), a metabolic myopathy caused by the deficiency of the lysosomal acid α-glucosidase. We showed that coincubation of Pompe fibroblasts with recombinant human α-glucosidase and the chaperone N-butyldeoxynojirimycin (NB-DNJ) resulted in more efficient correction of enzyme activity. The chaperone improved α-glucosidase delivery to lysosomes, enhanced enzyme maturation, and increased enzyme stability. Improved enzyme correction was also found in vivo in a mouse model of PD treated with coadministration of single infusions of recombinant human α-glucosidase and oral NB-DNJ. The enhancing effect of chaperones on recombinant enzymes was also observed in fibroblasts from another lysosomal disease, Fabry disease, treated with recombinant α-galactosidase A and the specific chaperone 1-deoxygalactonojirimycin (DGJ). These results have important clinical implications, as they demonstrate synergy between pharmacological chaperones and enzyme replacement. A synergistic effect of these treatments may result particularly useful in patients responding poorly to therapy and in tissues in which sufficient enzyme levels are difficult to obtain.