Plasma Of Normal Individuals Research Articles

Failure of ultracentrifugation as a means of separating plasma free insulin from immunoglobulin fraction prior to radioimmunoassay

It has been reported that ultracentrifugation of plasma will allow direct measurement of free insulin in the serum of diabetic subjects with insulin antibodies. To validate this method, we determined recovery of immunoreactive insulin and immunoglobulin G from the plasma of normal individuals after ultracentrifugation. The upper and middle fractions of plasma after ultracentrifugation were evaluated at several combinations of time and temperature (4°C, 25°C, or 37°C for 3, 4 or 5 hours). None of these conditions effectively removed all immunoglobulin G without causing concomitant loss of insulin. We conclude that ultracentrifugation of plasma prior to radioimmunoassay cannot be used to reliably determine free insulin concentration in the plasma from subjects with circulating insulin antibodies.

Low molecular weight C1q in systemic lupus erythematosus.

In sera of patients suffering from an exacerbation of systemic lupus erythematosus (SLE), increased amounts of abnormal C1q were detected, contrasting with decreased or even undetectable levels of normal C1q in these sera. When analyzed immunochemically by double immunodiffusion, this low m.w. C1q (LMW-C1q) appeared to be identical with the defective C1q in serum of individuals with an inherited, homozygous inability to produce functional plasma C1q. These persons show a tendency to develop SLE-like syndromes. Like the genetically defective C1q, the abnormal C1q molecule in SLE sera was hemolytically inactive, did not incorporate in C1, was found in the supernatant of euglobulin-precipitated serum, and appeared in the break-through fraction of a cation-exchange column. Sucrose gradients and gel filtration analyses supported the putative identity of the molecules. SDS-PAGE and immunoblots revealed the presence of subunits that reacted with antibodies against C1q and confirmed the C1q-like nature of LMW-C1q. Low levels of LMW-C1q were also detected in serum and plasma of normal individuals. A radial immunodiffusion technique was used to measure LMW-C1q in the serum of 54 patients. Although these patients were not selected for parameters of disease activity, their levels of LMW-C1q were significantly higher than those of normal individuals and children with decreased C3 levels due to acute glomerulonephritis.

Study of the Expression of a Glycoprotein of Retroviral Origin in the Plasma of Patients With Hematologic Disorders and in the Plasma of Normal Individuals

A 74,000 molecular weight glycoprotein was purified from the plasma of a patient with chronic myelogenous leukemia in blast crisis. Monoclonal antibodies were produced in the mouse and used to characterize this protein. It was shown to contain p15E antigenic determinants and portions of a reverse transcriptase. The level of this protein was found to be elevated in leukemic patients with high white blood cell counts and also in some patients with other hematopoietic disorders as compared to the level measured in normal individuals. The level of the protein was strongly reduced in acute leukemia patients after intense chemotherapy treatment. We tentatively conclude that this protein is of endogenous retroviral origin and perhaps regulates hematopoietic tissues.

Study of the expression of a glycoprotein of retroviral origin in the plasma of patients with hematologic disorders and in the plasma of normal individuals

A 74,000 molecular weight glycoprotein was purified from the plasma of a patient with chronic myelogenous leukemia in blast crisis. Monoclonal antibodies were produced in the mouse and used to characterize this protein. It was shown to contain p15E antigenic determinants and portions of a reverse transcriptase. The level of this protein was found to be elevated in leukemic patients with high white blood cell counts and also in some patients with other hematopoietic disorders as compared to the level measured in normal individuals. The level of the protein was strongly reduced in acute leukemia patients after intense chemotherapy treatment. We tentatively conclude that this protein is of endogenous retroviral origin and perhaps regulates hematopoietic tissues.

Lipid binding properties of the Tangier apolipoprotein A-I and its isoproteins.

The apolipoprotein A-I was isolated from the plasma of normal individuals and of three homozygous patients with Tangier disease by immunoprecipitation. The apoA-I isoforms were further fractionated by isofocusing on polyacrylamide gels. The physicochemical behavior of normal and Tangier apoA-I and of the isoproteins-2 and -4 was studied by monitoring the tryptophanyl fluorescence emission as a function of temperature, pH, and under exposure to guanidinium (guanidine) hydrochloride (GdmCl). Lipid-apoprotein complexes were generated by incubation with dimyristoylphosphatidylcholine and isolated by density gradient ultracentrifugation. Our results show that normal apoA-I and its isoprotein-4 associate with lipids to yield a complex containing 150-200 mol lecithin/mol apoA-I. The isoprotein-2 of normal apoA-I and the isoprotein-4 of Tangier apoA-I generate lipid-rich complexes with lecithin, while the isoprotein-2 of Tangier apoA-I shows only a limited association with lipids. ApoA-I normal and Tangier and their isoproteins-4 undergo a structural transition around 45 degrees C, which is not observed in the lecithin-apoA-I complexes. This transition is accompanied by an increased exposure of the tryptophanyl residues to the solvent. This transition was observed for the isoprotein-2 of apoA-I Tangier both in its lipid-free form and in the presence of lecithin. The pH denaturation of apoA-I and of the isoprotein-4 between pH 9 and 13 and between pH 7 and 2 is accompanied by a similar conformational transition. The transition occurs around pH 10.8 for the native apoproteins and is shifted towards respectively higher and lower pH's as result of the protective action of lipid binding on the protein conformation. Such an effect was not observed with the isoprotein-2 of apoA-I Tangier which is denatured at lower pH's both in its native form and in a lipid-protein mixture. Finally the denaturation of apoA-I by GdmCl indicates that apoA-I normal and Tangier undergo structural changes around 1 M GdmCl, whereas the apoA-I-Tangier-lecithin complex is more susceptible to denaturation than the complex with apoA-I normal. These data suggest that the apoA-I normal and Tangier and their isoproteins-4 are able to associate with lipids although the association between apoA-I Tangier with lecithin is weaker than that of apoA-I normal. The isoprotein-2 of normal apoA-I associates to a greater extent with lipids than the isoprotein-2 of Tangier apoA-I, whose structure differs from that of the isoprotein-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrone sulfate concentrations in plasma of normal individuals, postmenopausal women with breast cancer, and men with cirrhosis.

Estrone sulfate is quantitatively the most important estrogen in plasma. A method for its determination in human plasma is described, and the precision, accuracy, sensitivity, and specificity are defined. Free steroids were extracted from plasma with diethyl ether and steroid sulfates were isolated with use of Vlitos' reagent (methylene blue in dilute H2SO4/Na2SO4 solution). After enzymic hydrolysis, estrone was isolated by chromatography on Celite and measured by radioimmunoassay. The mean concentrations (nmol/L +/- 1 SD) of estrone sulfate were 2.51 +/- 0.90 for plasma from 13 women in follicular phase, 5.33 +/- 1.55 for 17 women in luteal phase, 0.89 +/- 0.60 for 44 postmenopausal women, and 0.96 +/- 0.43 for 24 postmenopausal women with breast cancer. Results for postmenopausal women with or without breast cancer did not differ significantly. For 13 normal men, estrone sulfate concentrations were 2.62 +/- 0.79 nmol/L, and for a group of 19 cirrhotic men the mean value was 1.43 +/- 0.95 nmol/L, significantly lower than normal.

The optical properties of alpha 2-macroglobulin from normal and from cystic fibrosis plasma.

The alpha 2-macroglobulin (alpha 2-M) was purified from the plasma of normal individuals and from that of cystic fibrosis patients. The proteins exhibited identical optical properties. Both proteins have an absorbance coefficient of A = 1060 g . cm-2 at 280 nm. The circular dichroism spectra are identical and indicate about 45% beta-sheet structure and almost no alpha-helix. The spectra of solutions at pH 8.0 do not change when trypsin is added. The fluorescence spectra of the alpha 2-M measured at pH 8.0 have contributions by tyrosine and tryptophan residues. The fluorescence intensities are identical and are enhanced about 30% when trypsin is added in 2:1 molar ratios.

Renal metabolism of beta 2-microglobulin. Experimental animal studies.

beta 2-microglobulin (beta 2M) is a protein of 11,800 daltons which occurs in the plasma of normal individuals at concentrations of approximately 2 microgram/ml. It is presumed to be relatively freely filterable. More than 99% of the filtered beta 2M is taken up by an active reabsorptive mechanism and catabolized by the renal tubule. The data presented here demonstrate that renal extraction is only slightly diminished by complete ureteral obstruction. The renal extraction of beta 2M is greater than can be accounted for by filtration alone. These data indicate that some uptake of beta 2M occurs from the peritubular capillary circulation. The loading of animals with beta 2M is associated with a marked tubular proteinuria suggesting that this protein may play a part in inducing tubular injury.

Prothrombin, thrombin and prothrombin fragments in plasma of normal individuals and of patients with laboratory evidence of disseminated intravascular coagulation

With specific immunoassays for prothrombin, thrombin-anti-thrombin-III complex and for prethrombin-1 antigen, we measured the levels of these proteins in the plasma of normal individuals and patients suspected of having chronic disseminated intravascular coagulation (DIC). The normal level of prothrombin in plasma was found to be 110–212 mg/liter, and of thrombin-anti-thrombin-III complex and prethrombin-1 antigen approximately 1 μg/ml. In patients with suspected DIC, prothrombin levels, as measured by immunoassay and two-stage clotting assay, were decreased in 14 of 19 studied. Levels of thrombin-anti-thrombin-III complex were normal in all the patients and those of prethrombin-1 elevated in only two of 19. The lack of elevation of these products is probably due to the small amount of prothrombin activated in these patients. Decreased synthesis may account for the low levels of prothrombin observed in some. Measurement of the level of these products of prothrombin is therefore not useful in the diagnosis and management of patients with DIC.

Studies on the protein defect in Tangier disease. Isolation and characterization of an abnormal high density lipoprotein.

High density lipoproteins (d 1.063-1.210 g/ml) were isolated from the plasma of normal individuals (HDL) and seven homozygous patients with Tangier disease (HDLt). In Tangier patients, the concentration of protein in the high density region (HDLt) was only 0.5-4.5% of normal. Immunochemical studies, including mixing experiments conducted in vivo and in vitro, indicated that HDLt was different from HDL. HDLt was the only high density lipoprotein detectable in the plasma of Tangier homozygotes. In heterozygotes both HDL and HDLt were present. HDLt was not detected in the plasma of over 300 normal persons and 10 patients with secondary high density lipoprotein deficiency and appeared to be a unique marker for Tangier disease.ApoHDL contained two major apoproteins designated apoLp-Gln-I and apoLp-Gln-II; together they comprised 85-90% of the total protein content. Both of the major HDL apoproteins were present in apoHDLt; but apoLp-Gln-I was disproportionately decreased with respect to apoLp-Gln-II, the ratio of their concentrations being 1: 12 in apoHDLt as compared with 3: 1 in apoHDL. Several minor apoprotein components which together comprise 5-15% of apoHDL were present in approximately normal proportions in apoHDLt. In the HDL of Tangier patients it was estimated that, compared with normal individuals, the concentration of apoLp-Gln-I was decreased about 600-fold and the concentration of apoLp-Gln-II about 17-fold. The decrease in these apoproteins was not due to preferential segregation with the lipoprotein fractions of d < 1.063 g/ml or with the plasma proteins of d > 1.21 g/ml. Tangier apoLp-Gln-I and apoLp-Gln-II appeared to be immunochemically identical with their normal counterparts, and no differences between the two sets of apoproteins were detected on polyacrylamide gel electrophoresis at pH 9.4 or 2.9. These results are most compatible with the hypothesis that the hereditary defect in Tangier disease is a mutation in an allele-regulating synthesis of apoLp-Gln-I.

In Vitro Inhibition of Plaque and Rosette Formation by α Globulin

Abstract Recent studies on the α globulin fraction from plasma of normal individuals have shown that it possesses immunosuppressive properties and interferes with lymphocyte functions in vitro. Cooperband et al. (1, 2) have shown that human α globulin inhibits lymphocyte transformation in vitro following phytohemagglutinin (PHA) and specific antigenic stimulation. Since α globulin fraction can also suppress antibody production against sheep red blood cells (SRBC) in vivo (3–6), experiments were conducted to assess its biologic activity in an in vitro system by the hemolytic plaque assay and the rosette formation technique. Materials and Methods. Alpha globulin was isolated from normal human plasma by DEAE cellulose chromatography according to the procedure described by Mannick and Schmid (7). Immunoelectrophoresis and acrylamide gel electrophoresis showed this material to be primarily α2 globulin. Male local adult white rats of a strain developed at the Hebrew University were used in the hemolytic plaque formation and the rosette formation experiments.

The occurrence of substances in human plasma capable of inducing the enzyme delta-aminolevulinate synthetase in liver cells.

We have demonstrated the presence, in the plasma of several patients with acute intermittent porphyria, of a substance which strongly induced the synthesis of porphyrins in chick embryo liver cells growing in primary culture. The induction response evoked by this humoral agent was in all respects similar to that elicited by drugs and hormones which are known to enhance porphyrin production by inducing the de novo formation of delta-aminolevulinate synthetase, the rate-limiting enzyme in this pathway. Inducing properties were not found in the plasma of normal individuals or in that from porphyric patients in remission. Significant inducing activity was, however, found in the plasma of some normal subjects ingesting drugs or contraceptive steroid mixtures. The occurrence of a potent inducer of delta-aminolevulinate synthetase in the plasma of certain porphyric patients may have clinical significance for these genetically susceptible individuals. It will also permit chemical characterization of those humoral agents which may be related to the episodic exacerbations of this hereditary liver disease.

Binding characteristics of transcortin in human plasma in normal individuals, pregnancy and liver disease.

SUMMARY The binding characteristics of transcortin have been measured in 16 normal individuals, 10 pregnant women, five subjects taking oral contraceptives or on oestrogen therapy and in six patients with advanced liver disease. The affinity of transcortin for cortisol as measured by its equilibrium constant was found not to differ significantly between the normal individuals and the rest. The transcortin capacity was greatly increased in pregnancy and by oestrogen therapy but was normal in the cirrhotics studied. Despite the increase in total plasma cortisol in pregnancy, the unbound plasma cortisol concentration was only slightly raised and no increase was noted in the oestrogen-treated group. One of the 'normal' group was found to be transcortin deficient with low total plasma cortisol but the concentration of unbound cortisol and the rate of cortisol secretion were normal.

Release of factor 8 (antihaemophilic factor) from perfused organs and tissues.

IT has been known for many years that patients with classic haemophilia bleed because they lack a plasma factor required for normal blood coagulation. This factor, factor VIII (antihaemophilic factor) of normal plasma, has been extensively investigated, but its exact site of origin is unknown. Recent developments in organ transplantation have stimulated us to look for the specific organ source of this procoagulant. If the site of synthesis of factor VIII could be identified, permanent replacement of the deficient factor in haemophilia might be possible. In the past, a variety of experimental approaches has been used to investigate the body's mechanisms for maintaining haemostatic levels of factor VIII: organ ablation1, total body irradiation2, administration of hepatotoxins3,4, reticulo-endothelial blockade5, physical exercise6, hormone administration7, and thromboplastin injections4. Several recent investigations have suggested that the spleen may play a part in the regulation of concentrations of factor VIII in the plasma of normal individuals. Weaver et al. demonstrated that factor VIII is maintained at normal concentrations when intact normal dogs are crosscirculated with haemophilic dogs, but that the concentrations decrease if a splenectomy is first performed on the normal animals8. In humans, Libre et al.9 have observed that splenectomy abolishes the rise in factor VIII that is known to follow injection of adrenaline into normal subjects10. Pool was able to recover antihaemophilic activity in extracts of splenic tissue, but not from a variety of other tissues11.

Distribution of Ammonia in Cells and Plasma

Abstract A method for the determination of plasma and whole blood ammonia is outlined which incorporates the desirable features of several older methods. By means of this method it has been demonstrated that the distribution of ammonia in erythrocytes and plasma of normal individuals is nearly equal to that of hydrogen ions. The ratio remains constant in cirrhosis, alkalemia, and ammonemia. It was observed in 82 consecutive admissions of cirrhotic patients that alkalemia was present in only a small percentage of the cases.

Diagnostic dissection by isoenzymes

Alterations to plasma and serum enzymes represent subtle protein modifications associated with disease states. It appears that these enzyme activities are associated with two or more closely-related multimolecular forms which are, nevertheless, chemically, physically and immunologically distinctv-, These components, isoenzymes, can be measured individually. I t is the purpose of this presentation to report on the isoenzyme composition oflactic dehydrogenase (LDH) in the tissues and plasma ofnormal individuals and of patients with various disease states.

The inhibitory action of human plasma on dinitrophenol uncoupling of oxidative phosphorylation

A factor from human plasma has been found and purified about 35-fold which will prevent the uncoupling action of dinitrophenol on the oxidative phosphorylation of rat liver mitochondria. This substance has the properties of a protein, but as yet has not been specifically identified. There appears to be more of this substance present in the plasma of normal individuals than in the plasma of patients with malignant disease.

The total carbonate content of the arterial and venous plasma in normal individuals

In the course of some observations on the respiratory disturbances seen in certain diseased conditions it became necessary for the sake of comparison to determine the total carbon dioxide of the arterial and venous plasma in individuals with normal cardio-respiratory mechanisms. In all the bloods of nineteen normal individuals at rest have been examined. In each case samples of arterial and venous blood were obtained within a few minutes of each other. Method.—The arterial blood was obtained by direct puncture of the radial artery using a technique similar to that employed by Stadie. The venous blood was collected without stasis from one of the large veins at the bend of the elbow. To avoid contact with air both samples were carefully delivered under albolene into paraffin coated centrifuge tubes and immediately centrifuged at high speed. One c.c. of the separated plasma was delivered under carbonate free ammonia water contained in a receiving cup of the Van Slyke apparatus and the total Co2 content determined by the method devised by Van Slyke.1 The method adopted was considered to be more advantageous for my purpose than the widely used method of first exposing plasma to 5.5 per cent. of CO2 and ascertaining the CO2 combining power. Results: The results presented in the accompanying table indicate that the total CO2 content of the arterial plasma is a fairly constant figure, averaging fifty-six volumes per cent. The venous plasma is always a little higher than the arterial in individual cases, the discrepancy being from three to eight volumes per cent. This discrepancy has been found to increase if the individual is allowed to take some light exercise, such as walking, just before the blood samples are taken.