Albert A. Dietz

Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis

Abstract The disc electrophoresis method of Davis (16) does not permit the separation of all 5 isoenzymes of LDH. The method was modified by omitting the stacking and sample gels, and substituting 40% sucrose solution. All 5 isoenzyme fractions then migrate into the separating gel. The material staining at the surface of the separating gel is shown to be “nothing” dehydrogenase. By accurately marking the distance the albumin-dye or the dye front is to migrate, excellent reproducibility of the isoenzyme separation can be achieved. The separations are such that there is no doubt as to the identification of the individual bands, and quantitation can readily be achieved.

Relation of vitamin E and serum lipids

Abstract Hyperlipemia of diverse etiology is well correlated with elevation of serum vitamin E level. When the various serum lipid fractions were measured, total lipid (r = 0.85) and β-lipoprotein (r = 0.81) gave the best correlations with the vitamin E level. Correlation of serum carotene with the various lipid classes gave lower values. A patient with fat-induced hyperlipemia had enormous elevations of serum vitamin E (up to 27.8 mg/100 ml); almost all of it was found in the chylomicron fraction. This patient provides evidence for Pelkonens hypothesis: namely, vitamin E is first absorbed in the chylomicron fraction and is later transferred to the (3-lipoprotein fraction, where it is almost exclusively found in normal sera in the post-absorptive state.

Disc electrophoresis of lactate dehydrogenase isoenzymes

Abstract The original method of disc electrophoresis permitted the localization of only 4 of the 5 lactate dehydrogenase isoenzymes. Conjecture as to the location of one of these is now settled in that it is demonstrated that the lactate dehydrogenase-5 does not migrate through the stacking gel. This was shown for human serum and liver. All 5 isoenzymes migrate into the separating gel by our earlier method. To obtain desirable separations of these for quantitation, it is necessary to use an upper gel of the same composition as the separating gel, to control the geometry of the gels, and to apply an optimal amount of sucrose with the sample.

Genetic control of lactate dehydrogenase and malate dehydrogenase isozymes in cultures of lymphocytes and granulocytes: Effect of addition of phytohemagglutinin, actinomycin D or puromycin

1. 1. The effect of the addition of phytohemagglutinin to cultures of purified lymphocytes and granulocytes upon the isozymes of lactate dehydrogenase (l-lactate:NAD oxidoreductase, EC 1.1.1.27) and malate dehydrogenase (l-malate:NAD oxidoreductase, EC 1.1.1.37) was studied. 2. 2. Increase in the proportion of muscle-type (M-type) lactate dehydrogenase was demonstrable with acrylamide-gel disc electrophoresis in 4 h, while a significant increase (P < 0.001) occurred within 24 h. Actinomycin D or puromycin prevented this change. Granulocytes, which had a high proportion of M-type lactate dehydrogenase initially, showed no alteration in the isozyme pattern in cultures with or without phytohemagglutinin. 3. 3. Two malate dehydrogenase isozyme bands were found in lymphocytes and granulocytes. The slow-moving Band 2 represented 44.8% of the total malate dehydrogenase in lymphocytes and only 13.4% in granulocytes. In cultures, with or without phytohemagglutinin, little change was seen in lymphocytes. Granulocytes, however, showed a marked increase in Band 2 (P < 0.001) in 24 h, but this occurred with or without the addition of phytohemagglutinin. Actinomycin D or puromycin blocked this change. 4. 4. The results indicate that in lymphocytes phytohemagglutinin acts at the two genetic loci controlling synthesis of muscle- and heart-type (M- and H-)lactate dehydrogenase polypeptide subunits. The synthesis of the two isozymes of malate dehydrogenase also may be under the control of separate genes which in granulocytes are influenced by conditions of cell culture, rather than by phytohemagglutinin.

Hemoglobin and haptoglobin determination by disc electrophoresis

Summary 1. An acrylamide disc electrophoretic method is described for separation of hemoglobins with direct quantitation by densitometry; the method is rapid, accurate, and convenient. 2. By this method, measurement of hemoglobin A 2 in 70 normal subjects gave a mean of 2.96%, range 2.1–3.9%. Twenty-one subjects with β -thalassemia trait gave a mean of 6.32%, range 4.4–8.4%. 3. Hemoglobin F can be separated and quantitated by densitometry when present as more than 10% of the total. By this method, values for hemoglobin F are higher than those obtained by alkaline denaturation. 4. The method also allows convenient determination of haptoglobin types and semi-quantitation of serum haptoglobin level.

Correlation of activities of the phosphatase of the urine and serum of normal and cirrhotic persons

Abstract The urinary excretion of acid and alkaline phosphatase in normal and cirrhotic men was correlated with the serum activities of these enzymes. Both serum and urine alkaline phosphatases were frequently elevated in cirrhosis and related to the severity of the disease. In normal men there was an inverse correlation between the serum activity of alkaline phosphatase and its clearance, suggesting the serum as a source. The increased excretion in cirrhosis could be due to changes in renal function or to renal damage. The activity of serum acid phosphatase is relatively constant in normal men whereas the urinary excretion is variable. In patients with cirrhosis, the urinary acid phosphatase activity is less variable and is in the low range. It is unlikely that the serum and the urine acid phosphatases are from the same source.

Dual action of pancuronium on succinylcholine block.

SummaryThe effects of pretreatment with both sub-paralyzing and paralyzing doses of pancuronium and d-tubocurarine, on the onset and duration of succinylcholine-induced neuromuscular blockade were evaluated and compared in 225 patients. D-tubocurarine antagonized both onset and duration of succinylcholine block, while pancuronium produced a dual effect, antagonizing the onset and potentiating the duration of succinylcholine block. Pretreatment with d-tubocurarine (0.07 mg/kg, 0.3 mg/kg and 0.6 mg/kg) increased the time to onset of succinylcholine paralysis from 28 to 118 per cent, and decreased the duration from 16 to 37 per cent. Pancuronium (0.02 mg/kg, 0.04 mg/kg and 0.08 mg/kg) also antagonized the onset of succinylcholine paralysis with increases of 32 to 114 per cent, but potentiated its duration from 30 to 103 per cent compared with succinylcholine alone in the same patients. Although pancuronium markedly inhibited serum cholinesterasein vitro (I50 = 5 x 10-7 mol) there was only a 10 per cent inhibition of cholinesterasein vivo after pancuronium 0.0S mg/kg.RésuméLes effets ďune injection préalable de d-Tubocurarine et de Pancuronium (à doses paralysantes et à doses inférieures ) sur la rapidité ďaction et sur la durée du bloc neuro-musculaire produit par la Succinylcholine, ont fait ľobjet de cette étude qui a porté sur 225 patients.La d-Tubocurarine retardait ľapparition et raccourcissait la durée ďaction du bloc à la Succinylcholine. Le Pancuronium avait un double effet, à savoir: un retard à ľapparition du bloc à la Succinylcholine et une potentialisation de sa durée.Des doses de 0.3, de 0.6 et de 0.7 mg/kilo en d-tubocurarine, injectées avant une dose de Succinylcholine retardaient ľapparition de paralysie à la Succinylcholine de 28 à 118 pour cent et diminuaient la durée de son effet de 16 à 37 pour cent.Le Pancuronium à des doses de 0.02, 0.04 et 0.08 mg/kilo retardait également ľapparition de la paralysie à la Succinylcholine de 32 à 114 pour cent mais augmentait sa durée de 30 à 103 pour cent.Bien que le Pancuronium montrait une inhibition marquée de la cholinestérase sérique,in vitro, on ne retrouvait qu’une diminution de 10 pour cent,in vivo, ceci après une dose de 0.08 mg/kilo.

Comparison of an electrophoretic and a chemical method for the estimation of serum lactate dehydrogenase isoenzymes

Summary 1. 2. For many clinical purposes, the chemical procedure will give results as useful as those obtained by electrophoretic fractionation, while being both simple and rapid.