Gaucher disease (type 1): Physical and kinetic properties of liposomal and soluble ‘acid’ β-glucosidase

Acid -glucosidase of human spleen, from either normal controls or patients with type 1 (adult) Gaucher disease, was incorporated into phosphatidylcholine liposomes. The non-incorporated (soluble) Gaucherenzyme had a higher apparent molecular weight than had the corresponding control. Liposomal acid -glucosidase prepared from Gaucher-spleen was more thermostable than was the corresponding normal enzyme; it was also stimulated by acidic lipids to a much lesser extent. The results suggest that the genetic mutation in type 1 (adult) Gaucher disease has multiple effects on the glycoprotein form of acid -glucosidase.     

Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency

Summary The biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK Mosul (homozygote), PK Bukarest_1,2, PK Hamburg₁, PK Köln₁, and PK Essen (compound heterozygote). PK Mosul showed normal positive cooperative substrate binding, PK Bukarest_1,2 exhibited noncooperative behavior, and PK Hamburg₁ and PK Köln₁ displayed mixed cooperativity, whereas PK Essen was negative cooperative. PK Mosul was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK Bukarest_1,2 a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK Bukarest₁). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK Bukarest₂). Direct sequencing showed the heterozygosity of the patient's mother (PK Bukarest₁/normal) at position 721 and of the patient's father (PK Bukarest₂ /normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK Hamburg₁ and PK Köln₁. The second mutation in these variants was not detected. In PK Essen no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.Supported by theDeutsche Forschungsgemeinschaft, Grants no. La 527/1 and Ne 416/1.     

Garrod's foresight; our hindsight

Archibald Edward Garrod introduced a paradigm, new for its day, in medicine: Biochemistry is dynamic and different from the static nature of organic chemistry. It led him to think about metabolic pathways and to recognize that variation in Mendelian heredity could explain an inborn error of metabolism. At the time, Garrod had no idea about the nature of a gene. Genes are now well understood, genomes are being described for one organism after another (including H. sapiens) and it is understood that genomes speak biochemistry (not phenotype). Accordingly, in the era of genomics, biochemistry and physiology become the bases of functional genomics and it is possible to appreciate why nothing in biology makes sense without evolution (and nothing in medicine will make sense without biology). Mendelian, biochemical and molecular genetics together have revealed what lies behind the four canonical inborn errors described by Garrod (albinism, alkaptonuria, cystinuria and pentosuria). Both older and newer ideas in genetics, new tools for applying them, and renewed respect for the clinician-scientist will enhance our understanding of the human biological variation that accounts for variant states of health and overt disease; an unsimple phenotype (phenylketonuria) is used to illustrate in some detail. What can be known and what ought to be done with knowledge about human genetics to benefit individuals, families and communities (society) is both opportunity and challenge.     

Capsular and ‘O’ serotype determinants of Bacteroides fragilis

Summary Forty-one strains ofBacteroides fragilis, 20 strains of otherBacteroides species and 14 strains of other genera were examined by the indirect immunofluorescent assay (IFA) using anticapsular serum. The sixty-oneBacteroides strains were O serotyped by direct agglutination tests using absorbed antisera raised against 23 strains, each with a different O antigenic determinant. All 41B. fragilis strains tested were positive by IFA with the anticapsular serum, but apart from one strain ofB. distasonis, none of the remaining 19 strains of other bacteroides, i. e.B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. melaninogenicus group andB. ureolyticus, and none of the 14 other bacterial species examined were positive. The majority of strains of saccharolytic bacteroides tested reacted with one of the 23 O antisera and were designated as a specific O serotype; a fewBacteroides strains had multiple agglutination reactions indicating the presence of multiple antigenic determinants. All O serotypes gave positive IFA tests with their homologous O antisera. Common capsular determinants and O antigenic determinants appear to exist on the same strains ofB. fragilis. Serological typing ofB. fragilis and related species would be useful in epidemiological studies.

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Kapsel- und O-Determinanten von Bacteroides fragilis

Zusammenfassung 41 Stämme vonBacteroides fragilis, 20 Stämme andererBacteroides-Spezies und 14 Stämme anderer Genera wurden unter Verwendung von Kapsel-Antiserum mit dem indirekten Immunfluoreszenztest (IFA) untersucht. Die O-Serotypisierung der 61Bacteroides-Stämme erfolgte mit dem indirekten Agglutinationstest; dabei wurden absorbierte Antiseren gegen 23 Stämme verwendet, von denen jeder eine unterschiedliche O-Determinante aufwies. Alle untersuchten 41 Stämme vonB. fragilis waren im IFA mit Kapsel-Antiseren positiv; hingegen war mit Ausnahme eines Stammes vonB. distasonis keiner der übrigen Stämme andererBacteroides-Spezies positiv, das heißt der GruppeB. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. melaninogenicus undB. ureolyticus; von den anderen geprüften 14 Bakterienspezies war ebenfalls keine positiv. Die Mehrzahl der Stämme der untersuchten saccharolytischenBacteroides reagierte mit einem der 23 O-Antiseren und wurde einem spezifischen O-Serotyp zugeordnet; einigeBacteroides-Stämme wiesen mehrfache Agglutinations-reaktionen auf, was für das Vorliegen mehrerer Antigendeterminanten spricht. Bei denselben Stämmen vonB. fragilis scheinen gemeinsame Kapsel- und O-Antigendeterminanten vorzukommen. Für epidemiologische Untersuchungen dürfte die Serotypisierung vonB. fragilis und verwandten Spezies von Nutzen sein.

     

Altered thiol status in patients with rheumatoid arthritis

Summary The thiol status of patients with rheumatoid arthritis is significantly different from that of controls. Plasma thiol levels are lower, albumin thiol reactivity is altered and intracellular thiol levels measured after hemoglobin precipitation are increased. These variations correlate with other indices of disease severity and are one measure of a disturbance in the degree of oxidation of the blood. Penicillamine, in common with other effective therapeutic agents, produces an increase in serum thiol concentration. It causes a greater effect on serum thiol reactivity than other drugs and in particular it increases fast reacting thiol levels without significantly altering the slow reacting thiol level.     

Mechanical properties and imaging characteristics of remanufactured intravascular ultrasound catheters

Intravascular ultrasound (IVUS) as a routine device in interventional cardiology is handicapped by its high price. 19 factory-made, remanufactured IVUS catheters which consist of sterilized, used phased-array IVUS transducers inserted into a new catheter shaft were compared with 23 new IVUS catheters. 3 mechanical and 4 imaging characteristics were assessed on a 5 point scale (1 = unacceptable, 5 = excellent). Mechanical as well as imaging properties of remanufactured IVUS catheter were comparable to new catheters with excellent ratings for each of the evaluated characteristics in 38 to 94% of remanufactured catheters and 50 to 96% of new catheters. The initial failure rate for remanufactured IVUS catheters was 31.6% vs. 4.3% for new catheters (P < 0.05). Overall failure rate was 47.3% for remanufactured catheters vs. 8.7% for new catheters (P < 0.05). The failure was due to an electronic connecting problem occurring during mechanical stress to the IVUS catheter. In conclusion, remanufactured IVUS catheters offer mechanical and imaging characteristics which are comparable to new catheters. Improvements in the remanufacturing process to resolve the high rate of electronic connecting problems may make this a promising approach to substantially lower the price of IVUS catheters.     

Enzyme replacement and beyond

During the last decade, enzyme replacement therapy for lysosomal storage diseases became a reality with the demonstration of its safety and effectiveness in type 1 Gaucher disease. Currently, enzyme replacement and several other potential therapeutic strategies are being developed for selected lysosomal storage diseases, including Fabry disease due to the deficient activity of -galactosidase A (-Gal A). The development and clinical evaluation of these new therapies require a stepwise process, each step being rigorously reviewed and approved by national or international regulatory agencies. For lethal disorders that affect small populations, such as many inherited metabolic diseases, this process can be accelerated by orphan drug and fast track regulations. As an example of the drug development process, the development of recombinant human -Gal A (r-hGal A) replacement for Fabry disease is presented, including the preclinical studies in the Fabry mouse model, and the clinical phase 1/2, phase 3, and phase 3 extension studies, which demonstrate the safety and efficacy of this new therapy.     

Changes in the activity of ‘active’ pyruvate dehydrogenase complex in the newborn of normal and diabetic rats

Summary At birth, hepatic active and dichloracetate-activated pyruvate dehydrogenase complex activities in the newborn of normal, mildly diabetic, and severely diabetic rats were similar. The active and dichloracetate-activated pyruvate dehydrogenase complex activities increased significantly during the first 2 and 6 postnatal h, respectively in the three groups of neonates (p<0.05). The greatest increase in both active and dichloroacetate-activated pyruvate dehydrogenase complex activity was observed in the neonates of mildly diabetic rats. Administration of glucose or insulin at birth to the newborn of normal rats caused a significant increase in the percentage of active pyruvate dehydrogenase complex activity within 1 h (p<0.01). Similar treatment caused no significant increases in the newborn of severely diabetic rats. The transient increases in active pyruvate dehydrogenase complex activity in the neonates of normal and diabetic rats were consistent with rapid disappearance of blood lactate during the first hours of postnatal life.     

Tris discriminates between the different α-glucosidase activities from extracts of human neutrophils

Summary In an attempt to detect acid maltase deficiency in neutrophils from patients with type II glycogenosis, without interference from the renal -glucosidase activity present in these cells, we have evaluated the contribution of the renal component in the total activity measured at pH 4.0 in extracts of human neutrophils. The renal contribution is about 13–25% and renal glucosidase appears to be closely related to the enzyme present on the epithelium of small intestine, which is known to be inhibited by Tris. We have used this compound as a selective inhibitor of the renal component of -glucosidase activity measured at pH 4.0 in total extracts of neutrophils. Our results demonstrate that 0.1 mol/L Tris is an inhibitor of the renal -glucosidase present in neutrophils and can be used to reduce the interference from this enzyme in assays of acid maltase.     

Computer simulation of the propagation of contrast medium in a coronary artery during one cardiac cycle

In some angiographic methods for measurement of mean coronary flow in ml/min, a threshold is applied to concentration-distance curves obtained from a constant rate injection by computing the intravascular contrast medium concentration along the main coronary branches. If the shape of the velocity profile would remain parabolic throughout the cardiac cycle, the correct threshold value would be 50% of the concentration at the injection site. But, coronary flow being strongly pulsatile, the shape of the velocity profile must be expected to vary appreciably within the cardiac phase. In order to investigate if a single, appropriate threshold value nevertheless exists for a great variety of coronary flow pulses and velocity profiles, the spreading of contrast medium injected continuously in a tube perfused by a time varying flow Q(t) was studied by computer simulation. While the particular time courses of flow and velocity profile appear to be of secondary importance, the ratio injection rate to peak coronary flow has a major impact. If it is equal to or greater than 1, a threshold value of 47% is the best choice. If the ratio is markedly less than 1, no appropriate threshold exists and use of the 47% threshold will result in considerable flow underestimations. This was fully confirmed by measurements of absolute coronary flow performed in patients.     

CD4+ and CD8+ subsets: Naive and memory cells in the peripheral blood of patients with systemic sclerosis

Summary Systemic Sclerosis (SSc; scleroderma) is associated with several immunological abnormalities, including altered proportion between lymphocyte subsets. Peripheral blood lymphocyte subsets from 25 patients with SSc were studied by two-colour flow cytometry using monoclonal antibodies against CD45RA and CD29 markers, which allow a dissection of CD4+ and CD8+ populations into naive and memory subsets. A decrease of the percentage of CD8+ (p<0.05) and of CD8+ CD29+ (p<0.001) cells was observed compared to that in 20 age and sex-matched controls. These abnormalities were not significantly associated with the extension of cutaneous disease or other clinical features of SSc nor with treatment, pattern of autoantibodies or HLA phenotype.     

Twenty-four hour profiles of plasma C-peptide in Type 1 (insulin-dependent) diabetic children

Summary Twenty-four hour profiles of plasma C-peptide an index of endogenous insulin secretion, were performed in 15 Type 1 (insulin-dependent) diabetic children. Plasma C-peptide was detectable in six children, of whom four (C-peptide producers) had peak values above normal fasting levels. In each of the six children with residual B cell function, there was a close correlation between plasma C-peptide and simultaneous blood glucose (r> 0.50, p< 0.05). Post-breakfast peak blood glucose was 10.2 ± 1.7 mmol/l (mean ±SEM) in the C-peptide producers and 18.7 ± 1.7 mmol/l in the 11 children with low or no detectable C-peptide. Mean M-value, an index of deviation from an ideal blood glucose, was lower in the C-peptide producers (p<0.05). It is concluded that residual functioning B cells in diabetic children behave physiologically in that insulin secretion fluctuates in accordance with the prevailing blood glucose; and that the pattern of action of injected insulin is more critical in non-C-peptide producers who lack the post-prandial dampening effect provided by residual endogenous insulin secretion.     

Glucagon immunoreactivity in plasma from normal and dystrophic mice

Summary The present investigation was undertaken to determine and characterize glucagon immunoreactivity in plasma from normal NMRI mice and from dystrophic mice and their unaffected littermates of the 129/ReJ strain. Very young dystrophic mice (6 weeks old) displayed much higher basal levels of plasma glucagon immunoreactivity than normal mice. In contrast, plasma concentrations of insulin and glucose were lower in these dystrophic mice than in normal NMRI mice. The plasma glucagon levels declined with age in both strains during the time-period studied (1.5–5 months). Gel filtration of plasma from dystrophic as well as normal mice on Sephadex G-200 revealed that a large part of the total glucagon immunoreactivity was eluted in fractions containing the immunoglobulins. The amount of the true glucagon part was lower in plasma from normal mice (about 0.2 g/l) than in plasma from mice of the dystrophic strain (0.4–0.5 g/l)). This finding was indirectly corroborated by the observation that a large intravenous glucose load decreased plasma glucagon by approximately 0.2 g/l in the non-dystrophic NMRI strain and by about 0.4–0.6 g/l in the dystrophic strain. Thus, the ability of glucose to suppress glucagon secretion appeared unaffected in the dystrophic mice. Glucose-induced insulin release, however, was considerably impaired in these animals. It is concluded that mice of the dystrophic 129/ReJ strain have higher plasma levels of true glucagon than mice of the non-dystrophic NMRI strain. Whether the abnormally high plasma glucagon levels in the dystrophic strain, particularly in very young dystrophic mice, might contribute to the development of the muscular dystrophy remains to be elucidated.     

Differences of cellular composition and adhesion molecule expression in “leukemic” as compared with “normal” human long-term bone marrow cultures

Summary Human long-term bone marrow cultures (HLTBMCs) were established with bone marrow samples collected from 15 patients with acute myeloid leukemia (AML) and compared with HLTBMCs from eight healthy volunteers. During 6 weeks of culture, the cellular composition of HLTBMCs was quantitatively studied. The cells of the HLTBMCs were divided into three main categories: fibroblasts, macrophages, and other cells (endothelial cells, hematopoietic cells and undefined cells). HLTBMCs derived from healthy volunteers demonstrated a very consistent development. The number of fibroblasts increased during culture and the number of macrophages decreased, resulting in a steady state after 3 weeks of culture. In contrast, HLTBMCs derived from patients with AML showed a strikingly different pattern of irregular development and a steady state was not reached under our conditions. The APAAP technique was used to demonstrate expression of adhesion molecules. VLA2, VLA5, VLA6, LFA1, Mac1, p150/95, ₂-chain, HCAM, ICAM1, NCAM, and VCAM1 were more expressed on normal as compared with leukemic bone marrow stromal cells, although this reached significance only for ₂-chain and NCAM. VLA1, 3, and 4 were expressed in a higher percentage on leukemic stroma (not significant). More expression was seen on normal as opposed to leukemic macrophages for the adhesion molecules tested, except for VLA5. The differences reached significance for the majority of molecules tested. It is concluded that striking differences exist in cellular composition and adhesion molecule expression between HLTBMCs from healthy individuals and those from patients with AML. This may have an impact on the pathogenesis of AML.     

Study of porcine and human isophane (NPH) insulins in normal subjects

Summary The plasma glucose, C-peptide and insulin responses to subcutaneously administered highly purified porcine, semi-synthetic and biosynthetic human isophane (NPH) insulin and diluting medium as control in normal male subjects were evaluated. Porcine and semi-synthetic human NPH insulins were administered at two dose levels of 0.15 and 0.30 U/kg body weight and biosynthetic human NPH at 0.15 U/kg body weight only. At the low dose level the three insulin preparations resulted in a similar maximal hypoglycaemic effect within 3–5 h after administration. However, over the remainder of the 11 h post-injection period, the plasma glucose level was lower after semi-synthetic human insulin. In contrast, at the 0.30 U/kg dose level, there was no difference in the early or late hypoglycaemic response between porcine and semi-synthetic human NPH insulins of equivalent pharmaceutical formulation. The clinical relevance of these findings needs further evaluation. The data suggest that for the intermediate-acting NPH insulin preparations, both the species of insulin, nature and quantity of the retarding protein and their subsequent interaction may determine their time-action characteristics.     

Contractile activity and prostacyclin generation in isolated coronary arteries from diabetic dogs

Summary The present study was aimed at determining the generation of prostacyclin (PGI₂)-like-material in coronary arteries from normal and diabetic (pancreatectomized) dogs as well as the contractile responses to prostacyclin of preparations from normal, diabetic and insulin-treated diabetic animals. PGI₂ produced a dose-dependent relaxation of coronary arteries from normal dogs. In contrast, those from diabetic animals were not relaxed; indeed, at low concentrations PGI₂ failed to evoke any effect but at higher ones it induced a distinct contraction. In arteries from diabetic animals treated with insulin, PGI₂ induced a biphasic contractile effect, which lay between that of normal controls and untreated diabetics. In addition the basal generation of PGI₂-like-material by coronary arteries was significantly higher in the diabetic (141±0.2 pg/mg, mean±SEM) than in normal dogs (59±0.2 pg/mg). The present experiments demonstrate that the generation of PGI₂-like-substance is significantly increased in coronary arteries from diabetic dogs, but the same vessels are unable to respond to added authentic PGI₂ with relaxation; on the contrary they react with a distinct positive contractile response.     

Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment

Summary Glomerular basement membrane thickness (GBMT) has been measured in streptozotocin diabetic rats treated with insulin. The study included 3 groups of 8 rats each: 1) a well-controlled group of diabetic rats under insulin treatment with a plasma glucose level reasonable close to normal values, 2) a poorly-controlled group also under insulin treatment with constant high plasma glucose values, and 3) an age and weight matched non-diabetic control group. After 6 months of diabetes, GBMT was measured applying an intercept method on 3 glomerular cross sections from each of the 24 animals. The measurements showed that mean GBMT was 132.2. nm in the non-diabetic control rats and 131.6 nm in the well-controlled diabetic rats. In the poorly-control-led group the mean GBMT was 140.4 nm, i.e. statistically significantly increased when compared to each of the two other groups, 2p=0.022 and 0.012 respectively.The results demonstrate that good blood glucose control in rats preserves normal GBMT.     

Gastric carcinoma and familial adenomatous polyposis (FAP)

We report two cases of FAP associated with gastric carcinoma. The literature on this subject, with special reference to fundic gland polyposis is reviewed.     

Pharmacokinetics of intravenous glibenclamide investigated by a high performance liquid Chromatographic assay

Summary The relationship between blood glucose and glycosylated haemoglobin (HbA_1c) has been investigated during an 8 week period in 53 Type 1 (insulin-dependent) diabetic women studied during the third trimester of pregnancy. Blood glucose estimations (fasting and 2 h post-prandially) were made an average of 41 times in each patient during this period and HbA_1c was determined once at the end of the study. There was a significant correlation between both the mean blood glucose over the preceding 8 weeks and the standard deviation of the fasting blood glucose with HbA_1c (r=0.69, p<0.001; r=0.46, p<0.001, respectively). A glycosylation index was calculated for each patient (HbA_1c divided by the mean blood glucose value). There was a significant correlation between the glycosylation index and duration of diabetes (r=0.68, p<0.001). In contrast, there was no correlation between red cell 2,3-diphosphoglycerate and HbA_1c or glycosylation index. These findings suggest that increasing duration of diabetes influences the post-translational formation of HbA_1c and that isolated HbA_1c values need to be interpreted with caution in the pregnant diabetic.     

Incidence of diabetes amongst people aged 18–50 years in nine British towns: A collaborative study

Summary The incidence of diabetes among people aged 18–50 years has been studied prospectively in nine towns, chosen to encompass the range of socioeconomic conditions and spread of latitude in England and Wales. The incidence of Type 1 (insulindependent) diabetes in this age group varied little between the towns. However, the incidence of Type 2 (non-insulin-dependent) diabetes was markedly higher in the towns with worse socio-economic conditions. The mean incidences were 23 per 100000 for the three worse towns and 10 per 100000 for the three better towns. This was not explicable by an association between the disease and social class. This finding has to be reconciled with known and hypothesised influences in the aetiology of Type 2 diabetes.This study was carried out in collaboration with Dr. P. I. Adnitt, Chester Royal Infirmary; D. L. Alexander, North Staffordshire Hospital Centre, Stoke-on-Trent; Dr. J. P. Bingle, District Hospital, York; Dr. B. F. Brearley, Sharoe Green Hospital, Preston; Dr. J. L. Day, The Ipswich Hospital; Dr. R. Fletcher, Clayton Hospital, Wakefield; Dr. K. J. Gurling and Dr. H. L. Matthews, Derbyshire Royal Infirmary; Dr. H. J. Lloyd, Royal Gwent Hospital, Newport; Dr. K. R. Hunter, Freedom Fields Hospital, Plymouth.