Complications in the determination of HDL2/HDL3 ratios

We investigated different procedures to subfractionate high density lipoprotein (HDL) into its density subfractions (HDL₂ and HDL₃). When pore-limit as well as zone polyacrylamide gel electrophoresis was used, it proved to be necessary to use freshly prepared and plasma protein-free HDL preparations. For a quantitative subfractionation of HDL in HDL₂ and HDL₃ we also tested density gradient ultracentrifugation. To avoid interference with LDL contamination a low starting density was required. Both methods, electrophoresis as well as density gradient ultracentrifugation were too laborious and time-consuming for large scale use. We devised a combination of both ultracentrifugation and electrophoresis as a method which is relatively appropriate for determinations of HDL₂/HDL₃ ratios on large scale.     

The role of lecithin : cholesterol acyltransferase in high density lipoprotein3/high density lipoprotein2 interconversion

Serum was incubated in vitro with and without inhibition of lecithin : cholesterol acyltransferase (LCAT, EC 2.3.1.43). High density lipoprotein₂ (HDL₂) and high density lipoprotein₃ (HDL₃) were separated by zonal ultracentrifugation and analysed for lipid and apoprotein contents. The incubation of fresh sera resulted in a time-dependent decrease in HDL₃ and an increase in HDL₂. At the end of 24 h incubation HDL₃ disappeared completely and the HDL₂ peak had reached its maximum. The newly formed HDL₂ was relatively enriched in total protein (apoprotein A-I, C-apoproteins) and cholesteryl esters, and depleted in phosphatidylcholine. Its migration in polyacrylamide gel electrophoresis was identical with HDL₂ contained in fresh serum or HDL₂ isolated from fresh serum by zonal ultracentrifugation. The generated HDL₂ particles exhibited the same electron microscopical characteristics as reference HDL₂ samples prior to incubation.Addition of Ellman's reagent to the incubation mixture or heat inactivation of the samples prior to incubation resulted in a complete inhibition of HDL₃/HDL₂ interconversion, whereas addition of 1 mol/l NaCl had no detectable influence. There was also a substantial increase in HDL₂ when VLDL-deficient serum was incubated at 37°C. Similarly, in fresh serum from a patient affected with familial lipoprotein lipase deficiency, HDL₃ was completely converted to HDL₂.Our experiments demonstrate that LCAT promotes HDL₃/HDL₂ interconversion in native serum irrespective of the presence or absence of triglyceride-rich lipoproteins and lipoprotein lipase.     

Serum vitamin E,lipid peroxide and glutathione peroxidase in patients with chronic pancreatitis

It has been reported that lipid peroxidation increases in patients with antioxidant deficiencies, such as vitamin E and glutathione peroxidase. The relationships between serum lipid peroxide and vitamin E on the one hand and glutathione peroxidase on the other were examined in 22 patients with chronic pancreatitis, often accompanied by malabsorption of fats and fat-soluble vitamins due to the impaired exocrine pancreatic function.Both serum vitamin E concentrations and glutathione peroxidase activities were depressed, especially in patients with chronic calcifying pancreatitis. On the other hand, serum lipid peroxide levels were elevated. A significant negative correlation was found between the serum lipid peroxide levels and vitamin E concentration.These findings suggest that an elevation of the serum lipid peroxide level may be due to the lack of an antioxidative defense mechanism, such as vitamin E, against lipid peroxide.     

Erythrocyte transketolase activity in uremia

We performed a study concerning the activity of erythrocyte transketolase and thiamine metabolism in 73 uremic patients with or without neuropathy and 67 normal control subjects. Although the total vitamin B1 level in whole blood was high in the uremic patients, the transketolase activity in the hemolysate and the thiamine pyrophosphate effect on it were lower than those of normal subjects. The values of the transketolase activity of the two groups were statistically correlated with the levels of the thiamine pyrophosphate effect and the vitamin B1 content of the blood. Inhibition of transketolase activity was apparent in the uremic patients. However, this inhibition did not seem to be the only cause for the development of uremic neuropathy since no significant difference in these activities was observed among uremic patients with and without neuropathy. Moreover, a direct correlation could not be confirmed between transketolase activity and motor nerve conduction velocity.     

Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients

The enzymic activities of erythrocyte phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and glutathione reductase (EC 1.6.4.2) have been measured in 54 primary gout patients, 35 individuals having hyperuricaemia and 51 healthy controls. Statistical analyses have shown a significant increase (p less than 0.01) in the enzymic activity of erythrocyte PRPP synthetase in both the hyperuricaemic and gout groups compared with the controls. No correlation between activity and age was found in any of the three clinical groups. A significant decrease (p congruent to 0.01) was found in the enzymic activity of red cell glutathione reductase in the gout group compared with the other two groups. The biochemical significance of the changes in enzymic activities of the two enzymes in primary gout is discussed.     

Improvement of the FeCl3 test for phenylpyruvic acid

A simple alternative colorimetric method has been developed for quantitative determination of phenylpyruvic acid in urine based on the improved stability of the ferric complex of enol-phenylpyruvate in water-dimethylsulfoxide 50%vol. mixed solvent. The method can be applied directly to urine or to a dry ether-extract of urine. Operating on the dry ether-extract of urine, detection of phenylpyruvic acid is possible at an amount of as low as 1 mg/ 100 ml. Whereas, if urine samples are processed, detection is possible for a concentration of as low as 5 mg/100 ml and quantitative determination for a concentration greater than 10 mg/100 ml. The test carried out on a diaper wet with urine retains the same sensitivity and shows instantaneous development of the green colour.     

The use of concanavalin a crossed immuno-affinoelectrophoresis to detect hormone-associated variations in α1-acid glycoprotein

α₁-Acid glycoprotein produces three peaks on crossed immuno-affinoelectrophoresis with concanavalin A in the first dimension, which is indicative of a degree of heterogeneity in the carbohydrate portion of this plasma glycoprotein. A different pattern, however, showing relatively less concanavalin A-binding material, is found in association with and after an increase in female sex hormone levels. Our observations strongly suggest a prolonged hormonal effect on the carbohydrate composition of a₁-acid glycoprotein, which may alter the metabolism or the function of the protein.     

A radiochemical method for the determination of transketolase activity in erythrocyte hemolysates

A radiochemical method for the determination of transketolase activity is reported. It is based on incubation of erythrocyte hemolysates with radioactive ribose 5-phosphate followed by isolation of sedoheptulose 7-phosphate on anion-exchange columns. The experimental conditions are discussed and the presence of phosphatase activity in the incubation mixture is demonstrated. The proposed method is compared with a current colorimetric method. Reference values are given for the transketolase activity and the thiamine pyrophosphate effect.     

The estimation of pyrophosphate in urine with uridine-5'-diphosphoglucose pyrophosphorylase.

The concentration of pyrophosphate in urine has been determined using the enzyme uridine-5'-diphosphoglucose pyrophosphorylase. The technique is relatively quick and easy to perform. Reproducibility of results was good, and results from recovery experiments were excellent. The mean concentration of pyrophosphate found in early morning urine samples from 14 normal adults was identical with that reported by other workers using an ion-exchange method to measure pyrophosphate. The pyrophosphate concentration was related approximately to the phosphate concentration.     

Determination of Nτ-methylimidazoleacetic acid (a histamine metabolite) in urine by gas chromatography using nitrogen-phosphorus detection

N^τ-Methylimidazoleacetic acid, the quantitatively most important metabolite of histamine, was isolated from urine by ion exchange chromatography. After esterification with 2-propanol and extraction, N^τ-methylimidazoleacetic acid was analyzed by capillary gas chromatography with nitrogen-phosphorus detection, using N^τ-ethylimidazoleacetic acid as internal standard. The synthesis of this internal standard is described. In contrast to the methods hitherto described, this method is appropriate for use in clinical chemical laboratories. Normal 24-h excretion ranged from 8.3 to 18.5 μmol (n = 20).Five patients with mastocytosis, a patient with chronic myelocytic leukemia and a patient after an anaphylactoid reaction on acetylsalicylic acid showed highly elevated values.     

Immunological similarity of NCA (non-specific cross-reacting antigen) in feces with alpha 1-acid glycoprotein

We have recently suggested that carcinoembryonic antigen (CEA) may contain alpha 1-acid glycoprotein (AG) antigenic determinant. In the present work we examined a protein with CEA-like activity in the feces of healthy subjects (NCA) for immunological cross-reactivity with AG. When the perchloric acid extract of feces was fractionated on a Sephadex G-200, two fractions (large and small molecular weight) were obtained. The large molecular weight fraction had higher CEA activity than the small one. The perchloric acid (PCA) extract of feces was subjected to affinity chromatography using anti-CEA bound to Sepharose, and the bound protein was labelled with 125I, and then fractionated on a Sephadex G-200 column. Two radioactive peaks, Peak 1 corresponding to an approximate Mr of 180 000 and Peak 2, corresponding to an approximate Mr of 60 000 were found. Both peaks showed immunoreactivity with either anti-CEA or anti-AG. This experiment suggests the presence of two kinds of CEA-reactive proteins in feces: one which may be a big protein with immunological similarity to AG and a second which appears to be a hydrolysed fragment of this protein.     

Pregnancy-specific beta 1-glycoprotein (SP1) determined by means of electroimmunoassay, radial immunodiffusion and nephelometry.

A comparison has been made between rocket-immunoelectrophoresis (RIE), radial immunodiffusion (RID) and automated immunonephelometry (AIP) in the assay of pregnancy-specific beta 1-glycoprotein (SP1) in serum from pregnant women. Using RIE an interaction was demonstrated between the various SP1-reactive molecular populations causing a bias of up to 10%. An interaction corresponding to this phenomenon cannot be demonstrated when using RID and AIP. When correlating the serum-SP1 concentration of samples containing various ratios of SP1-reactive molecules by means of RIE, RID and AIP, it was demonstrated that there was no correlation between the results achieved using one method compared to the results achieved by either of the other methods. The results achieved using one method can therefore exclusively be judged from reference values determined using the same method. The analysis time is essentially shorter with AIP than with RIE and RID.     

Reduction of hyperornithinemia with a low protein,low arginine diet and pyridoxine in patients with a deficiency of ornithine-ketoacid transaminase (OKT) activity and gyrate atrophy of the choroid and retina

Five patients with gyrate atrophy of the choroid and retina showed a 60% or greater decline in plasma ornithine levels during a five week trial of a low protein (10–15 g/day), low arginine (0.50–0.75 g/day) diet supplemented with essential amino acids and pyridoxine administration. These declines in plasma ornithine levels were seen in Patients 1–4 with the pyridoxine non-responsive variant and in Patient 5 with the pyridoxine responsive variant. No harmful systemic side effects were noted. Patients 1–4 continued on a modified low protein (20–35 g/day), low arginine (1.25–1.75 g/day) diet as tolerated and Patient 5 on pyridoxine alone. After one year no improvement was observed in visual acuities, visual fields, final dark adapted thresholds and full field electroretinograms for four patients. In contrast to the other four patients, Patient 3, with relatively poor control of plasma ornithine levels, showed signs of progression of the chorioretinal atrophy and further reduction of electroretinographic responses. Patients 1–4 continue on the dietary regimen and Patient 5 on pyridoxine alone to determine whether any lowering of plasma ornithine levels will modify the course of their ocular disease.     

The effect of surgery on the renal excretion of β2-microglobulin

Surgical trauma causes an increase in the renal excretion rate of β₂-microglobulin whilst creatinine excretion is not influenced.The increase in the renal excretion rate of β₂-microglobulin is probably the result of an increased release of β₂-microglobulin by the cells which exceeds a maximum in the active tubular reabsorption of the compound by the proximal tubule cell.The renal excretion of β₂-microglobulin is proportional to the relative clinical trauma score.     

Electrophoretic separation of Aγ and Gγ human globin chains in nonidet P-40

Electrophoresis in cellulose acetate in the presence of 3% Nonidet P-40 can resolve two neutral genetic variants, ^Aγ and ^Gγ human fetal globin chains. The ratio of these two chains, determined by densitometry of the electrophoretic strips, is in excellent agreement with the Gly-Ala ratio obtained by chemical analysis of the cyanogen bromide fragment 7CB3. It is suggested that the detergent binds preferentially to the hydrophobic amino acid segment 133–141 in the ^Aγ chain, thus masking either a Lys or an Arg residue at the two extremes.     

The microheterogeneities of thyroxine-binding globulin and alpha 1 protease inhibitor (alpha 1-antitrypsin) are interrelated

Addition of 125I-thyroxine to serum allows autoradiography for thyroxine-binding globulin microheterogeneity to be carried out after isoelectric focusing has been performed to display (by protein stain) the heterogeneous bands of the alpha 1-antitrypsin (PI) system. Comparison of the protein stain for PI with the autoradiograph for thyroxine-binding globulin indicates that these two systems are interrelated with the major bands of the PI system corresponding to the bands on the autoradiograph. This correspondence holds for PI variants other than the common M type and in particular it holds for the deficient Z type in which the autoradiograph for thyroxine-binding globulin is strikingly different from normal. We conclude that the major cause of microheterogeneity of TBG is due to an association with the PI system under the conditions of isoelectric focusing as normally performed. Precipitation experiments with antisera to PI and TBG suggest that the complex between these biologically important globulins may occur under conditions other than isoelectric focusing, but further work will be needed to examine this possibility.     

The origin of plasma creatine kinase 1, detected in patients during and following open heart surgery

Plasma creatine kinase 1 (CK-1) was detected intra-operatively in 6 out of 6 patients and postoperatively in 15 out of 22 patients, undergoing cardiac surgery. A transient increase in plasma levels of creatine kinase 2 (CK-2) and total creatine kinase (CK-tot.) activity was observed in all patients. The disappearance rates for the 2 isoenzymes in the circulation were CK-1: Kd = 4.7 X 10(-3) min-1, and CK-2: Kd = 0.60 X 10(-1) min-3. Analysis of vessel and heart tissue showed that the saphenous vein contained mainly CK-1; high activities of all three isoenzymes were found in the parts of the heart investigated. Most probably, both CK-1 and CK-2 are liberated from injured cardiac tissue.     

Continuous tissue ph and transcutaneous PO2 measurement as an index of tissue perfusion in critically ill patients

Measurements of transcutaneous PO₂ (tcPO₂) and tissue pH (pHt) were compared with arterial values in 28 critically ill patients. Eleven of the patients were undergoing continuous flow cardiopulmonary bypass and the other 17 were admitted to the Intensive Therapy Unit (ITU) suffering from a variety of medical and surgical disorders. A total of 135 observations were made on the 28 patients. The overall correlation between the two sets of measurements was poor. The correlation coefficient (r) between arterial PO₂ (Pa,O₂) and tcPO₂ was 0.41 (P < 0.01) and between arterial pH (pHa) and pHt 0.67 (P < 0.01). However, the value varied greatly in different groups of patients.In those patients with cardiopulmonary failure, the tCPO₂ and pHt measurements rapidly and accurately reflected changes in peripheral tissue perfusion, by contrast the core-peripheral temperature deficit did not change significantly. It is concluded that the technique can be used as a sensitive index of peripheral blood flow and may have prognostic significance.     

Hypocholesterolaemia in the rat after p-chlorophenoxyisobutyric acid (CPIB) administration as a synthetic triglyceride, 1,3-dipalmitoyl 2(p-chlorophenoxyisobutyroyl)-rac-glycerol

A synthetic triglyceride analogue, 1,3-dipalmitoyl $\text{2(p-}\text{chlorophenoxyisobutyroyl}\text{)-rac-}\text{glycerol}$, was fed to rats and its effect compared to that of clofibrate. Both compounds significantly lowered plasma cholesterol concentration and the decreases essentially paralleled each other, even when the dose of drug or the period of administration was varied. No consistent effect was noted on the plasma triglyceride concentration. The results suggest that synthetic lipids are potentially suitable carriers for the oral administration of pharmacologically active agents.     

Fast fluctuations of glycosylated hemoglobins. II. Hemoglobin A1c determination and oral glucose tolerance test

Hemoglobin A₁ (Hb A₁) levels were determined during glucose tolerance tests. A good correlation (r = 0.83) was observed between fasting Hb A₁ and the area of the hyperglycemia peak. During the tests the levels of Hb A₁ increased by about 10% of the initial value and about one third of this rapidly formed glycosylated hemoglobin remained stable after a 4 days storage period.