A preparative method for the isolation of genetic variant forms of glucosephosphate isomerase and a study of five variants

A method has been developed for the rapid, quantitative separation of normal and abnormal glucosephosphate isomerase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Five different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. While the physical and catalytic properties of most of the variants were similar, clear differences in the stability of the allozymes were apparent. In order to detect mutations affecting the stability, a series of different stability tests are required.     

The effects of near maximum exercise of serum enzymes: the exercise profile versus the cardiac profile

Serum lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) are elevated following exercise and myocardial infarct (MI) in man. Since the “Cardiac profile” of these enzymes is used for the assessment of the extent and duration of an MI, it is of interest to note the changes caused by exercise that may affect the Cardiac profile. Five trained healthy middle-aged males, all chronic joggers, were rested for four days, during which time blood samples were monitored daily to establish baseline values. A near-maximum run from 6 to 10 miles was performed, and bloods were sampled 1 h post exercise and at 8-h intervals for four more days during which time no other exercise was permitted. Enzyme analysis, performed on a SMAC, showed the following results: (1) all runners showed elevations in LDH and CPK; (2) LDH was maximum at 1–8 h post exercise, while CPK was maximum at 24 h. CPK also showed the greatest percent increase over baseline. (3) Isograms of LDH and CPK showed that the cardiac isozyme of LDH (LDH-1) increased the most (a “1–2 flip” was present), whereas the large increase in CPK was due to the skeletal muscle type (MM) alone. Thus, an “Exercise profile” of serum enzymes, distinct from that seen following MI, is produced following exercise even in well-trained individuals. These results render isozymal profiling a more certain diagnostic modality.     

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.     

Neuroma pain model: Correlation of motor behavior and body weight with autotomy in rats

A rat pain model was investigated by examining the correlation of autotomy (self-mutilation) score with motor behavior and body weight change after sciatic nerve transection, encapsulation and neuroma formation. Observations of motor behavior and body weight changes (e.g. feeding behavior) as an index of pain were considered to have several advantages over scoring the degree of autotomy. Motor activity of 14 rats (12 neuroma, 2 sham), measured using a stabilimeter, was compared on a weekly basis to autotomy scores for a total of 7 weeks after surgery. Additionally, body weight of 26 rats (20 neuroma, 6 sham surgery) was monitored for 4 weeks following surgery. While autotomy, changes in body weight and abnormalities in motor behavior were observed after surgery, no significant Spearman rank correlation coefficients were determined for any week and thus no significant relationships were found between autotomy score and motor activity or body weight. However, it was observed that rats after sham surgery gained significantly more weight than rats after sciatic nerve transection. Therefore, these results cast doubt on the validity of autotomy score as the sole index of pain.     

A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue

The oxidation of protoporphyrinogen to protoporphyrin, which is catalyzed by the enzyme protoporphyrinogen oxidase, is an important step in heme biosynthesis. We describe a fluorometric assay for protoporphyrinogen oxidase activity in mammalian tissues which measures the conversion of the non-fluorescent protoporphyrinogen to the fluorescent protoporphyrin. Using these assay conditions, the mean level of protoporphyrinogen oxidase activity in sonicated rat liver mitochondria was 12.3, and in sonicated human cultured skin fibroblasts was 1.97 nmol protoporphyrin/mg protein/h.     

Triglyceride-poor very low density lipoprotein in human serum.

The chemical and physical properties of very low density lipoproteins, isolated from the pool of the sera of 60 persons with high pre-beta and normal triglyceride and cholesterol concentrations, have been studied. These very low density lipoproteins, a designated as triglyceride-poor very low density lipoproteins, consist of 20.5% phospholipids, 30.8% free cholesterol, 15% cholesterol esters and 33.7% triglycerides. Their protein content consists of 54.5% apo B, 26% apo A, 11.5% apo E and only 8% apo C, so they differ from any serum lipoprotein described until now. Triglyceride-poor very low density lipoproteins consist of spherical particles 300-450 A in diameter as revealed by electron microscopy.     

The relationship between the cholesterol content and subfraction distribution of plasma high-density lipoproteins

High density lipoprotein subfractions (HDL₂ and HDL₃) were separated from the plasma of 25 healthy volunteers (13 males, 12 females) by rate zonal ultra-centrifugation. The rotor elution profile, measured at 280 nm, was used with the specific extinction coefficient for each subfraction (HDL₂, 0.60 ± 0.11 mg protein/A_280nm, HDL₃, 0.86 ± 0.10 mg protein/A_280nm(n = 25) to calculate their plasma concentration. Their protein and lipid composition were also determined by chemical analysis.Plasma lipids, lipoprotein cholesterol, apolipoprotein A-I, apolipoprotein A-II and apolipoprotein B levels were measured in the same subjects and correlated with the HDL subfraction concentrations. HDL cholesterol and apolipo-protein A-I concentrations correlated significantly (p< 0.01 and 0.02 respectively) with plasma HDL₂, but not with HDL₃. Indeed, the significantly higher levels of HDL cholesterol and apolipoprotein A-I in the female group could be attributed entirely to an increase in circulating HDL₂. This data supports the proposal that the latter subfraction is the major contributor to the anti-atherogenic role of plasma HDL.     

A new micro-assay for hyaluronidase activity.

A new micro-method for the assay of hyaluronidase activity is described. The method utilizes chondroitin sulfate as a substrate. The degraded products of chondroitin sulfate by hyaluronidase are determined by modified disc gel electrophoresis. The products are first concentrated into a single band in acrylamide gel, and then the band is stained with cetylpyridinium chloride. The absorbance of the band is proportional to the amount of the degraded products. The hyaluronidase activity is therefore linearly related to the absorbance. This procedure represents a sensitive method for the assay of hyaluronidase activity in the serum and urine.     

A new variant mucolipidosis: Biochemical investigations on two siblings

Biochemical studies are presented on two siblings with some features of Mucolipidosis III, but with distinctive clinical findings. Levels of β-galactosidase, -mannosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and -fucosidase found in serum from these patients ranged from 10 to 100 times higher than normal. The ratio of heat stable to heat labile serum isoenzymes of N-acetyl-β-glucosaminidase is considerably greater than normal.

An extremely low activity of β-galactosidase was found in fibroblasts cultured from one patient. Levels of the remaining enzymes were in the low normal range. Similarly, β-galactosidase levels were low in heart, kidney, liver, spleen and lung of one patient who died during the course of the study. Activities of the remaining enzymes were close to normal.

No excessive excretion of mucopolysaccharide was noted, however, changes in distribution of several fractions were found. Mucopolysaccharide labeled with radioactive sulfate was degraded by cultured fibroblasts at a normal rate.

In addition to clinical differences, the biochemical studies further demonstrate the uniqueness of these patients.     

GM1-ganglioside beta-galactosidase in leukocytes and cultured fibroblasts.

GM1-ganglioside hydrolysis by leukocytes and fibroblasts, tissues easily obtainable from patients, was investigated using 3H-labeled GM1 and was found to be at least as active as that reported for any other tissue. Sodium taurocholate was required for the reaction, the crude bile salt at an optimum concentration of 0.4% producing twice as much activity as pure taurocholate at its optimum concentration of 0.8%. Leukocyte GM1-ganglioside beta-galactosidase and 4-MU-beta-gal cleaving activities were similar, 134.5 +/- 23.3 and 179.8 +/- 25.4 nmol/h/mg protein, respectively. In cultured skin fibroblasts and amniotic fluid cells these enzyme activities were 4 to 5 times higher. Homozygotes for GM1-gangliosidosis showed negligible activity while in heterozygotes the leukocyte GM1-cleaving activity was reduced to one-third of control values. In leukocytes from patients with four other sphingolipid storage diseases the activity was either normal (Krabbe's, Tay-Sachs, Metachromatic leukodystrophy) or increased (adult Gaucher's).     

Tooth pulp-evoked potentials in the monkey: cortical surface and intracortical distribution

The distribution of tooth pulp-evoked potentials (TPEPs) was characterized in the primary motor (MI), primary somatosensory (SI) and secondary somatosensory (SII) cortices of the monkey. Bipolar electrical tooth pulp stimulation elicited TPEP components P23 and N44 over SI, P26 and N72 over MI, and P72, N161, P280, N420, P561 and N662 over SII. Muscular artifacts and extradental input did not affect the TPEP as demonstrated by experiments using a neuromuscular blocking agent and removal of the pulp, respectively. The short latency TPEPs recorded over SI and MI were evoked by low stimulus intensities and activation of A beta nerve fibers, whereas the long latency TPEPs recorded over SII required higher stimulus intensities and the additional recruitment of A delta nerve fibers. Intracortical recordings revealed polarity reversals of components P23 and N44 in area 3b, P26 and N72 in area 4, and P72, N161, P280, N420, P561 and N662 in the upper bank of the lateral sulcus (SII). Evidence presented in this study suggests that TPEPs recorded from SI and MI relate to non-nociceptive mechanisms while TPEPs recorded from SII relate to nociceptive mechanisms.     

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.     

A solid phase enzyme immunoassay for the quantitation of serum ferritin

A non-competitive solid phase enzyme immunoassay for the measurement of ferritin in human serum is described. This procedure involves the use of specific antibody covalently attached to derivatized hydrophilic microparticles and enzyme labeled specific antibody. Reproducible results were achieved within 4 h for ferritin in serum in the range of 4 ng/ml to 250 ng/ml. Ferritin levels as low as 0.4 ng/ml can be measured. The enzyme immunoassay and three commercially available radioimmunoassay (RIA) kits were used to determine serum ferritin levels in healthy adults. Good agreement was found between the enzyme immunoassay and the RIA methods.     

The contribution of drinking water lead to maternal blood lead concentrations

The association between domestic water lead concentrations and blood lead concentrations has been examined in 232 mothers at delivery. The blood lead was found to vary significantly with the cube root of the water lead. This association was stronger for first flush water lead rather than for running water lead. This study emphasises the danger to mothers and to their children of environmental lead over-exposure in areas of soft acid plumbosolvent water.     

A temperature conversion nomogram for glycosylated hemoglobin analysis

Analysis of glycosylated hemoglobins by commercial mini-column methods was shown to vary linearly with the ambient temperature. However, the slopes of the temperature dependence lines for diabetic and healthy individuals was different. A method for temperature correction and a nomogram is proposed.     

The separation of plasma lipoproteins using gel electrofocusing and polyacrylamide gradient gel electrophoresis.

Polyacrylamide gel electrofocusing and gradient electrophoresis have been used to separate the lipoproteins in whole plasma and in fractions prepared by sequential flotation in the ultracentrifuge and by precipitation with dextran sulphate and manganous chloride. After the two-dimensional separation, high density lipoproteins appear as a zone showing noticeable heterogeneity with respect to both isoelectric point and molecular weight. Low density lipoproteins are resolved as a compact spot while very low density lipoproteins are visible as a long horizontal streak across the top of the electrophoresis gel. The implications of the technique for the analysis of lipoprotein patterns in pathological plasmas are discussed.     

Connective tissue activation. XVII. Radioimmunoassay of a human platelet derived connective tissue activating peptide (CTAP-III) and specificities of anti-CTAP-III sera

The platelet-derived connective tissue activating peptide (CTAP-III) has been shown to be an important factor stimulating the metabolism and proliferation of human connective tissue cell strains, including synovial tissue cells. The quantities of CTAP-III affecting the cellular changes and the amounts in various biologic fluids and tissues are small. The objectives of this study were to develop a radioimmunoassay (RIA) for CTAP-III and to ascertain the specificities of the anti-CTAP-III sera reagents. The antisera were shown not to cross-react with a number of polypeptide hormones. However, two other platelet proteins, β-thromboglobulin and low affinity platelet factor-4, competed equally as well as CTAP-III for anti-CTAP-III antibodies in the RIA system. Thus, the three platelet proteins are similar or identical with respect to those portions of the molecules constituting the reactive antigenic determinants. The levels of material in normal human platelet-free plasma that inhibited anti-CTAP-III-12S!-CTAP-III complex formation were determined to be 34 ± 13 (S.D.) ng/ml.     

Serum angiotensin converting enzyme activity in patients with chronic renal failure on long term hemodialysis.

Serum angiotensin converting enzyme activity was assayed by a spectrofluorometric method in 19 patients with chronic renal failure on long term hemodialysis and 19 control subjects. Converting enzyme activity was increased in 11 of 19 (58%) patients compared with the controls (p less than 0.005). There was no correlation between serum converting enzyme activity and plasma renin activity or blood pressure in the patients. Possible mechanisms responsible for the increased converting enzyme activity are discussed.     

The application of gel filtration and specific analyses of urinary carbohydrate and protein material to the diagnosis of metabolic disorders.

A gel filtration method developed for urinary carbohydrate and proteinaceous material based on Bio-Gel P-2 cross-linked polyacrylamide gel has been coupled to specific continuous automated analyses for neutral and acidic carbohydrate and protein. The multichannel analytical system has been applied to urine from normal subjects and from known cases of genetic hyperglycosaminoglycanuria (“mucopolysaccharidosis”) to analyse not only the non-dialysable, high molecular weight material but also the low molecular weight material which comprises over 90% of urine solutes in many cases.Urine from affected patients was easily distinguished from that from normal subjects and the method also differentiated between various syndromes. For the Morquio syndrome with mucopolysacchariduria and Scheie syndrome the ratio of acidic to neutral carbohydrate (carbazole : cysteine ratio) in the high molecular weight material is the lowest (less than 0.6) whilst for the Hunter syndrome the value is greater than 2.0, with values for Hunter and Sanfilippo syndromes being intermediate. The ratio of high to low molecular weight material for the Morquio syndrome with mucopolysacchariduria in the case of the borate-carbazole assay is lower (below 0.2) but in the case of the L-cysteinesulphuric acid assay is higher (above 0.6) than values obtained for the other syndromes. There was evidence for a division within the Sanfilippo syndrome using this molecular size ratio for the L-cysteine-sulphuric acid assay, some with values greater than 0.5 and some with values below 0.3. In urine from cases of Morquio syndrome with mucopolysacchariduria there was also evidence of large amounts of intermediate molecular weight material.The application of this method to studies of other disorders of inherited metabolism is equally possible and is discussed.