Oscar Bodansky

[55] Lactic dehydrogenase (clinical aspects)

Publisher Summary This chapter discusses the clinical aspects of lactic dehydrogenase. Lactic dehydrogenase (LDH), like many other enzymes, is present in the urine, and its daily excretion has been reported to be increased in renal disease, particularly in cases of carcinoma of the kidney and bladder. It has been reported that 15% of a series of 38 patients with malignant neoplasms of the urinary tract had normal excretions and conversely, that approximately 70% of 63 patients with benign conditions had increased excretions of LDH. The assay most commonly used is based on the principle that involves the reduction of pyruvate to lactate and the concomitant oxidation of reduced diphosphopyridine nucleotide (DPNH) to DPN as observed at 340 mμ. For their determinations, a final volume of 3 ml is used in which the final concentrations of reactants were: pyruvate, 1 mM; DPNH, 0.23 mM; phosphate buffer, 33 mM (pH 7.3). Subsequent investigators, 16–22 whether they utilized this principle for assay of crystalline preparations of tissue lactic dehydrogenase activity, or introduced it for assay of lactic dehydrogenase in other body fluids, have varied these concentrations. The temperature has ranged from presumably room temperature (24–25°) to 38°. In the method used in our laboratory the final concentrations of pyruvate and DPNH are 0.33 mM and 0.1 mM, respectively, and the temperature is 37°.

Relationship of the electrophoretic patterns of phosphohexose isomerase and glutamic oxaloacetic transaminase in human tissues to the patterns in serum of patients with neoplastic disease

Abstract Ultracentrifugal fractionation of human and animal tissues indicated that about 90 per cent of phosphohexose isomerase (PHI) was localized in the high speed supernatant. Glutamic oxaloacetic transaminase (GOT) was about equally divided between the fraction sedimented at 24,000 × g for 10 minutes and the supernatant fraction after centrifugation at 105,000 × g for 60 minutes. Starch paste electrophoresis indicated that in thirty-five of forty tissue specimens and in serum from seven normal persons and from thirty-three of thirty-eight patients, the PHI was confined completely to a cathodic area in the gamma globulin region. In all tissues GOT was distributed into two main areas, one in the region of gamma globulin and the other in the alpha-beta globulin region. The GOT in serums of four normal persons and of twenty patients was completely confined to one peak, the alphabeta globulin region. Eleven patients had two electrophoretic regions of GOT activity, in the alpha-beta globulin region and the gamma globulin region. Six patients exhibited the usual area of activity in the alpha-beta globulin region and a second area in the albumin region. Four of these also had a third area of activity in the gamma globulin region. Previous work in the literature has indicated that the cathodic gamma globulin component of GOT is largely mitochondrial in origin. The appearance of this component in the serum in a number of cases appeared to be associated with an acute phase of the patients disease, and its disappearance with subsidence of this phase.

Interruption of Perfusion of Isolated Rabbit Heart upon Reaction of Coronary Flow.

We have not found described the effect of interruption of the perfusion of the isolated mammalian heart upon the reaction of the coronary flow immediately subsequent to resumption. This very simple experiment may be used to demonstrate one of the most fundamental chemical properties of muscle, increased acid production under conditions of inadequate oxygen supply. This increased acidity is demonstrable with the isolated rabbit heart, perfused with Ringer-Locke 1 solution. In some instances we have altered the formula, using CaCl2, 0.012% instead of 0.024%, or varying the NaHCO3 strength; the former change appears immaterial to the result, but reduction in NaHCO3 strength makes the reaction very distinct. In order that the perfusion fluid may wash the heart of blood and clear the tissue of accumulated diffusible acid products in unusual amount, perfusion is carried out for 20 or 30 minutes, when the beat has usually become fairly regular, and the fluid escaping from the heart nearly constant in reaction from minute to minute. The perfusion is then stopped by clamping off the tube leading from the Ringer reservoir, or by lowering of the perfusion pressure from the usual adequate value of 50 to 70 cm. of Ringer to about 4 cm. The latter procedure may readily be carried out with the apparatus used by us 2 by providing a rubber tube connection of adequate length for the Ringer reservoir. With zero or inadequate perfusion pressure so caused, the heart not infrequently shows at first a period of increased force of beat, but soon becomes irregular and finally stops at the end of usually 8 to 20 minutes. If the perfusion is now resumed with the former pressure, the fluid escaping from the heart is more acid than before for a period of 1 to 5 minutes or even longer, and not infrequently also temporarily increased in amount.

[99] Phosphohexose isomerase: Clinical aspects

Publisher Summary This chapter discusses the clinical aspects of phosphohexose isomerase (PHI). Elevations of this enzyme activity have been found in the serum of patients with cancer, hepatitis, myocardial infarction various myopathies, leukemia, and other diseases. Fructose reacts in solution with resorcinol and HCl at a temperature of about 80° to form a red-colored compound. This is the well known Seliwanoff reaction for ketose sugars and consists in the formation, first, of hydroxymethylfurfural and, secondly, in the condensation of this compound with resorcinol to form a cherry red colored product or products. Heating under the usual conditions of this reaction, 15 minutes at 80° in 6 N HCl, results in the hydrolysis of F-6-P to fructose and inorganic phosphate to the extent of 40–50%. Fructose and the unhydrolyzed F-6-P, reacts to the same extent with HCl and resorcinol on the basis of the fructose content. The method for PHI described is based on the conversion of G-6-P to F-6-P at pH 7.4 and 37°. The activity is defined as milliunits—that is, the millimicromoles of F-6-P formed per minute per milliliter of reaction mixture by 1 ml of serum or other biological fluid under the conditions described above. The calibration curve would ordinarily yield a value for the amount of F-6-P or fructose formed per milliliter of reaction mixture by 0.04 ml of serum in 30 minutes. The calibration curve may also be prepared so as to translate the absorbance readings directly into mill/units of activity by multiplying the F-6-P values on the abscissa by 1.00/0.04 × 1/30 or 0.833.

Use of commercially available preparations of fructose 6-phosphate in the determination of phosphohexose isomerase activity☆

Abstract The purity of six commercial preparations of fructose 6-phosphate has been studied. Each of the preparations was diluted, based on organic phosphorus content, to yield a 0.55 m M solution. The enzymically determined molarities of these solutions ranged from 0.13 to 0.51 m M . The ratio of the color intensity obtained by interaction of these preparations with resorcinol and HCl was compared to that obtained with an equimolar concentration of fructose. These ratios ranged from 0.47 to 0.89. Treatment of the fructose 6-phosphate samples with 1 N HCl for 180 min at 100°C liberated 55 to 91% of the phosphorus. The implications of these findings in the assay of phosphohexose isomerase activity are discussed. It is recommended that conversion of glucose 6-phosphate to fructose 6-phosphate, with colorimetric determination of the end product, be used in the assay of phosphohexose isomerase.

Effect of Parathormone on Bone Phosphatase Activity in vitro

The relationship between hormonal and enzymic activity is of considerable biological interest. Kay 1 noted high plasma phosphatase values in generalized ostitis fibrosa. Page 2 reported a decrease in the bone phosphatase in rats after parathormone injections. Bodansky and Jaffe 3 obtained increases in plasma phosphatase 8 hours after injection of single large doses in the dog and decreases in the guinea-pig after 1 and 2 days. Heymann 4 reported a direct effect of parathormone in vitro. He observed decreases of 50 to 100% in bone phosphatase activity when 0.1 units of parathormone were added per cc. of hydrolysing mixture. The effect on kidney and intestinal phosphatase was much less marked. He conducted his experiments at a pH of 7.7 using a 50% concentration of a phosphatase preparation which had been extracted from the tissue with glycine buffer. Sodium glycerophosphate and hexosephosphate were employed as substrates. The parathormone was added in a glycine solution. We have studied the effect of parathormone∗ on the phosphatase activity of aqueous extracts of rat and cattle bone. The sodium diethylbarbiturate buffer of Michaelis was used. This buffer in 0.5% concentration is without effect on phosphatase activity and hence is to be preferred to glycine which, in the concentrations used . for buffering, retards phosphatase activity about 30%. 5 The technique of tissue phosphatase activity estimation was similar to that used in previous studies. 5 0.0127 M sodium β glycerophosphate (Eastman Kodak) was used as the substrate. The reciprocal of the time in minutes necessary to liberate 0.05 mg. of inorganic phosphate as P per cc. of hydrolyzing mixture was used as the measure of enzyme activity (Q).

[261d] Clinical aspects of leucine aminopeptidase☆☆☆

Publisher Summary This chapter focuses on clinical aspects of Leucine Aminopeptidase. An enzyme closely related to or identical with leucine aminopeptidase is widely distributed in plants, microorganisms, and various animal tissues. These studies have been carried out with different substrates and often under varying conditions. The following values were obtained per milligram tissue in man: brain, 3.0; duodenum, 2.8; striated muscle, 2.7; kidney, 2.4; spleen, 2.3; liver 2.0. Low values, 0.3 to 2.0 units, were also obtained for other human tissues such as uterus, esophagus, thymus, aorta. Leucine aminopeptidase appears to exist in several molecular forms. The activity of leucine aminopeptidase in urine, in other body fluids, and even in tissues has also been investigated with regard to possible clinical applications. The average activity, expressed as milligrams of β-naphthylamine liberated in a 24-hour urine, under specified experimental conditions, was 82± 29 for 30 normal males and 43±12 for 50 normal females. Of 24 patients with cancer and normal serum leucine aminopeptidase activities, 10 had elevated urinary enzyme activities.

Reduction of Toxicity of 2-Sulfanilamidopyrimidine (Sulfadiazine) by Phenylazo-alpha-alpha-diaminopyridine Hydrochloride (Pyridium) in the Mouse

The possibility of antagonism in systemic toxicity between the sulfonamides and other compounds has begun to receive attention during the past year. McCarty 1 found that para-aminobenzoic acid has no effect on the acute fatal toxicity of sulfapyridine in mice. David, et al., 2 reported that pentobarbital exerted a protective action against the toxicity of intravenously administered heparin-sodium sulfapyridine mixtures in dogs. Richards 3 found that small doses of urethane or pentobarbital decrease the fatal toxicity of intravenously administered sodium sulfapyridine in rabbits and dogs. In the present paper it is shown that the acute toxicity of 2-sulfanilamidopyrimidine (sulfadiazine) in the mouse is reduced by phenylazo-alpha-alpha-diaminopyridine hydrochloride (pyridium). The toxicity of sulfanilamide is not affected by this compound. The percent mortality in a group of mice injected subcutaneously with a given dose of sulfonamide was compared, under the same experimental conditions, with the mortality in groups receiving this dose and, in addition, doses of 10, 30 or 100 mg of pyridium per kg. For each experiment, 3 groups consisting of 8 to 15 mice each were employed. A total of 269 mice was used in the experiments here reported. The sodium salts of sulfadiazine and sulfanilamide∗ were injected subcutaneously in concentrations of 5 to 10%. Pyridium was injected in a 0.2% solution or a 0.5% suspension. When both sulfonamide and pyridium were employed, they were injected within a few seconds of each other into widely separated sites. The mice were given water and food ad lib. and the number of deaths occurring in 4 days noted.

Phosphorus Components in the Blood of Normal and Rachitic Infants

Summary 1. The phosphorus of the blood was fractionated by a combination of acid and phosphatase hydrolysis and the results compared in normal and rachitic infants. 2. In infants with rickets there is a reduction in the acid soluble phosphorus of the blood which is made up of decreases in the inorganic phosphate, the readily acid hydrolysable phosphorus, and the fraction not hydrolysable by bone phosphatase under stated conditions. 3. The chemical nature of these fractions is discussed.

Subnormal Temperatures from Poisoning in Relation to Toxicity Determination

It seems important, especially in connection with toxicity determination, to point out that toxic doses of many substances may produce fall of temperature before death or recovery. An old clinical direction calls for note of the body temperature in case of poisoning and special arrangements if needed to reduce heat loss. Incidentally, the statement, “quinine has no significant effect on the normal temperature in man or in animals”, 1 appears to need revision, the statement, “in toxic doses the fall is proportional to the collapse”, 2 being truer. Quinine has produced subnormal temperatures in man, 3 and in the rabbit fall of 12°C., 4 and the related cinchonine fall of 14°C. in this animal. 5 We were led to note rectal temperatures in guinea pigs, used for preliminary toxicity determinations on cinchona bases, by unwittingly repeating an old observation, 6 finding live animals, sometimes quite active, cadaverously cold to the touch. The drugs were given subcutaneously in the flank, in doses mostly in the lethal range, which is for most of these bases 100–400 mg. per kg. Using an average of about 7 animals in each series, we found in the case of each of 22 cinchona bases at least one animal in each series showing fall of 6° C. or more before death or recovery. The phenomenon seems common to all members of the group, including quinine, and is not limited to the cinchona series, for we found it also with morphine and quinoline, and Louvier 7 has noted it with phenobarbital. The fall is not necessarily premortal: thus, one of our animals recovered completely after fall of 9°C. from hydrocinchonine; Simpson 8 reported recovery of a monkey after fall of 24.6°C. from ether anesthesia and chilling.