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Human tumor cells grown in fetal calf serum and human serum: influences on the tests for lymphocyte cytotoxicity, serum blocking and serum arming effects.

Peripheral blood lymphoid cells (PBL) from cancer patients and normal donors were tested against three melanoma cell lines grown in either 10% fetal calf serum (FCS) or 2.5-5% human AB serum in order to determine if the heterologous membrane (HM) antigen or other FCS antigens acquired from the bovine serum supplement could influence lymphoid cell-mediated cytotoxicity in vitro. FCS-grown melanoma cells were more susceptible than the AB serum-grown subline to lymphocyte cytotoxic effects. Arming effects by autologous sera on normal donor lymphocytes and to a lesser extent on lymphocytes of cancer patients were more pronounced on the FCS-grown M12 melanoma cells. This effect was abrogated when the cells were grown in human AB serum for at least 8 weeks. The non-HM tumor-associated antigen remained at the same original low level. Blocking effects were more evident on the AB-grown M14 melanoma line. These data suggest that the FCS antigens on the cell surface may have been responsible for the augmented PBL cytotoxicity. The anti-FCS antibody present in normal and cancer patients' blood induced an antibody-dependent cellular cytotoxicity (ADCC). Elimination of arming activity against HM or other FCS antigens from AB-grown cells may have made the serum blocking factors more apparent. However, cytotoxicity against tumor cells by PBL from normal donors was still apparent even on the human serum-grown cells, suggesting that a different antigen-antibody system was also responsible for this "non-specific" activity.

Animals

Total serum protein, serum protein fractions and serum immunoglobulins in colostrum-fed and colostrum-deprived calves.

Total serum protein levels, serum protein fraction levels, and specific serum immunoglobulin class or subclass levels were measured in colostrum-fed (CF) and colostrum-deprived (CD) calves during the first 144 hours after birth. Total serum protein values increased at 24 hours in the CF group and then decreased slightly at 144 hours. The increase in total serum protei5) in beta1-, beta2-, and gamma-globulins. The beta2- and gamma-globulin levels decreased by 144 hours, while the serum level of beta1-globulin continued to increase. The CD calves exhibited a significant decrease (P less than 0.05) in total serum protein at 24 hours, folhours, the level of beta1-globulin inlowed by a significant increase (P less than 0.05) at 144 hours. At 24 hours, the level of beta1-globulin decreased slightly, and the level of beta2- and gamma-globulins increased slightly. At 144 creased, and the level of gamma-globulin decreased. The beta2-globulin level did not change. At birth, immunoglobulin (Ig) M was detected in 5 of the 10 calves, IgG1 in 6 of the 10 calves, and IgG2 in 3 of the 10 calves. By 24 hours after birth, all CF calves had detectable levels of IgM, IgG1, and IgG2, and there were significant increases (P less than 0.01) in the mean serum levels of all 3 immunoglobulins. By 144 hours after birth, the serum levels of IgM, IgG1, and IgG2 decreased to various degrees. At 24 hours, the IgM level had not increased in CD calves; however, the level of IgG2 appeared to increase slightly, and the mean IgG1 level increased by approximately 50%. By 144 hours after birth, there was a significant increase (P less than 0.01) in the mean level of serum IgM. The level of IgG, also appeared to increase substantially, while the level of IgG2 appeared to increase slightly.

Animals

An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis.

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.

Acute Disease

Increased serum prolactin in diabetic ketoacidosis; correlation between serum sodium and serum prolacting concentration.

In 8 patients with diabetic ketoacidosis, serum prolactin was measured and found to be elevated (24.8 +/- 10.2 ng/ml, mean +/- SEM). After correction of the ketoacidosis, the prolactin level decreased to 10.9 +/- 6.4 ng/ml (normal range men 4.9 +/-0.8, women 5.1 +/- 1.6 ng/ml). The scatter of prolactin values was wide. A study of the correlation between the serum prolactin values and chemical parameters that are altered in diabetic ketoacidosis was therefore undertaken. Serum prolactin values did not correlate well with the serum bicarbonate concentration or serum osmolality. However, a significant negative correlation between log serum prolactin concentration and serum sodium concentration was demonstrated (r = -0.61, P less than 0.01). It is suggested that serum prolactin may possible participate in sodium retention in man as has been demonstrated in studies on animals.

Adult

Serum thyroxine, serum % -- T3 -- binding, and serum T3 in Zambians.

Serum total thyroxine (T4) was measured in clinically euthyroid and clinically hyperthyroid subjects, using the method of competitive protein binding. In 26 euthyroid subjects the mean +/- 1SD was 7.7 +/- 1.8 micrograms/dl and in 17 clinically hyperthyroid subjects, the mean +/- 1SD was 18.1 +/- 3.1 micrograms/dl. This difference is statistically significant. Serum % 125(1) -- T3 binding was measured using the principle of competition for 125(1) -- T3 between the unknown serum and the resin in the assay kit. In 18 clinically euthyroid subjects, the mean +/- 1SD was 112.6 +/- 4.4 % while in 14 clinically hyperthyroid subjects the % 125(1) -- T3 binding had a mean +/- 1SD of 71.4 +/- 11%. The difference is statistically significant. Serum total triiodothyronine was measured by radioimmunoassay. In 31 clinically euthyroid subjects the mean +/- 1SD was 146 +/- 19.8 ng/dl while in 19 clinically hyperthyroid sugjects the mean +/- 1SD was 390 +/- 111 ng/dl. This difference is statistically significant. There was a negative correlation which was not statistically significant between the serum T4 and % T3 binding. The measurements of these thyroid function indices were precise.

Humans

[The radio immune level of total serum oestriol during pregnancy. An examination of the method and comparison of the concentrations of total serum oestriol, non-conjugated serum oestriol and the excretion of total urinary oestrogens (author's transl)].

The authors report on a preliminary study of the level of total serum oestriol in the third trimester of pregnancy by the bundle method of estimating this with "oestriol 1251 RIA Kit" (Amersham, Buchler). The criteria on which the quality of the method depends, viz, sensitivity, specificity, reliability, and accuracy, together with its practicability are satisfactory. Correlations between total serum oestriol and free serum oestriol and total urinary oestrogens are good and correspond with those given in the literature. This method therefore can be used in clinical practice. All the same, it still has to be established which parameter better reflects the feto-placental state. Is it the free serum oestriol or the total serum oestriol?

Estriol

[Effect of chronic inhalation of carbon disulfide intoxication on the content of various lipid metabolism indices in rat serum, aorta and cardiac muscle. I. Total fats, triglycerides, free fatty acids in serum, free and esterified cholesterol in serum, aorta and cardiac muscle].

Fatty compounds were examined in blood serum, aorta and cardiac muscle of rats exposed to carbon disulphide in low (0.44 mg/l) and high (0.8 mg/l) concentration for 3 and 6 months in the toxicological chamber. The content of total lipids, triglycerides and free fatty acids in serum was determined using methods adopted in laboratory diagnostics. Separation of neutral fats in serum and tissue was done using thin--layer chromatography and free and esterified cholesterol was quantitatively determined by colorimetric methods. Statistical analysis of the results was based on the Student's test. In the rats intoxicated with CS2 increased content of total fats, triglycerides, free fatty acids in serum and cholesterol in cardiac muscle was found; these changes are not statistically significant. Statistically significant increase of free and esterified cholesterol was found in serum and aorta of the rats intoxicated with CS2. These changes were found mostly in rats intoxicated for 3 and 6 months with low concentrations of CS2.

Animals

Serum dependence of expression of the transformed phenotype: experiments with subline of mouse L fibroblasts adapted to growth in serum-free medium.

Characteristics of LSF cells grown in serum-containing and serum-free medium were compared. LSF is a subline of the L-strain of mouse transformed fibroblasts adapted to continuous growth is serum-free medium. Proliferation of LSF cells in monolayer on solid substratum was almost similar in serum-containing and in serum-free media. However, several other characters were found to be altered by the addition of serum to the serum-free medium: ability of cells to form colonies in semi-solid medium increased considerably; agglutinability of cells by Concanavalin A increased; uptake of 2-deoxy-D-glucose by the cells increased considerably; ability of cells to metabolize benzo (a) pyrene was inhibited; cell morphology was altered and, in particular, the cells became less spread on the substratum and density of microvilli on the cell surface increased. All these changes induced by serum were reversed by transfer of the cells back into serum-free medium. Thus, addition of serum increased the expression of a number of cellular traits characteristic of transformed phenotype, while in serum-free medium partial phenotypic reversion of transformation was observed. A possible role of serum in the expression of the transformed phenotype is discussed. It is pointed out that cell lines adapted to growth in serum-free medium provide an experimental system convenient for analysis of the effects of different serum components on the cell phenotype.

Animals

The significance of serum progesterone and serum unconjugated oestradiol-17beta in unaborted hydatidiform mole.

Both serum progesterone and serum unconjugated oestradiol-17beta (Oe2) were measured by competitive protein binding assay and radioimmunoassay respectively in 42 cases of unaborted hydatidiform mole. Serum human chorionic gonadotrophin (HCG) was measured by a haemagglutination-inhibition technique. In 26 cases of intact molar pregnancies without theca lutein cysts (TLC), serum progesterone ranged from 18.0 to 289.0 ng/ml with a mean +/- standard error of the mean (SEM) of 65.9 +/- 13.1 ng/ml; serum Oe2 ranged from 4.0 to 37.0 ng/ml with a mean +/- SEM of 17.9 +/- 1.9 ng/ml; serum HCG ranged from 60 to 1920 IU/ml with a mean +/- SEM of 531.5 +/- 105.7 IU/ml. In contrast, 16 cases of intact molar pregnancies with TLC had serum progesterone ranging from 34.1 to 288.0 ng/ml with a mean +/- SEM of 134.1 +/- 2.4 ng/ml; serum Oe2 ranging from 1.7 to 76.3 ng/ml with a mean +/- SEM of 31.5 +/- 5.3 ng/ml; serum HCG ranging from 320 to 2560 IU/ml with a mean +/- SEM 1400 +/- 196.2 ng/ml. The differences between the mean of these three hormones in hydatidiform mole with and without TLC were significant (progesterone: P less than 0.005; Oe2: P less than 0,0125; HCG: P less than 0.005). There was a significant correlation between serum HCG and serum Oe2 (coefficient of correlation r = +0.3565, P less than 0.0125) and between serum Oe2 and serum progesterone (r = +0.3787, P less than 0.0125). There was no significant difference in the mean levels of serum progesterone, Oe2 and HCG in hydatidiform mole with and without subsequent malignant sequelae. The mean ratios of Oe2/progesterone were essentially similar in moles with and without TLC and with and without malignant sequelae. The significance of these findings are discussed.

Choriocarcinoma

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

Animals

Circadian rhythms of serum renin activity and serum corticosterone, prolactin, and aldosterone concentrations in the male rat on normal and low-sodium diets.

To investigate the control of aldosterone secretion, non-stress levels of serum aldosterone, corticosterone, and prolactin, and renin activity were determined at 4-h intervals during 24-h light-dark cycles in adult male rats on regular and low-sodium diets. Circadian rhythms of plasma aldosterone, prolactin, and corticosterone concentrations and of serum renin activity were demonstrated during a regular sodium diet. When the rats were on a low-sodium diet, a circadian rhythm of serum corticosterone and aldosterone concentration was observed, but there was no circadian variation in serum renin activity or in serum prolactin concentration. Serum aldosterone concentration correlated with serum corticosterone concentration (r = 0.48) and serum renin activity (r = 0.36) during a low-sodium diet. Serum prolactin concentration did not correlate with serum aldosterone concentration or serum osmolality. These data are compatible with a role for renin and ACTH, but not for prolactin, in the modulation of aldosterone secretion in the rat.

Aldosterone

Immunochemical investigations on lipopolysaccharides and acidic polysaccharides from serum-sensitive and serum-resistant strains of Escherichia coli isolated from urinary-tract infections.

Factors that may determine the variable resistance of urinary strains of Escherichia coli to the bactericidal activity of normal human serum have been analysed. No statistically significant difference was found in the amount of lipopolysaccharide (LPS) that could be extracted from serum-sensitive and serum-resistant strains by either the phenol-water or warm-saline techniques. The ratio of LPS O-side-chain sugars to core sugars was not found to be significantly greater in serum-resistant than in serum-sensitive strains. A sugar resembling D-glycero-D-mannoheptose was found in LPS from some of the strains; in one case the sugar was shown to be associated with the O-side chain moiety. Lipopolysaccharides from all but two of the strains contained the E. coli R1 core structure. No consistent difference was observed between serum-sensitive and serum-resistant strains in either the amount of acidic polysaccharide extracted or its red-cell agglutination-inhibiting activity; nor was a clear relationship found between sensitivity to serum and sensitivity to R-specific bacteriophages. It is concluded that no one mechanism of serum resistance explains the response to serum of the E. coli strains examined in this study.

Blood Bactericidal Activity

Serum testosterone compared with serum zinc in man.

Serum zinc was measured in 154 male and 95 female blood donors. Men had higher serum zinc levels than women, the difference being statistically significant (p less than 0.001). The correlations between serum zinc and serum testosterone, serum zinc and serum FSH and serum zinc and serum LH were studied in 40 men aged 28-60 years and a statistically significant positive correlation was found only between serum zinc and serum testosterone in men of age 36-60 years (p less than 0.005). These results do not support the theory that slight zinc deficiency primarily effects pituitary gonadotropins but indicate that it would rather act on testicular level.

Adult

Low serum triiodothyronine and high serum reverse triiodothyronine in old age: an effect of disease not age.

Serum concentrations of T4, T3, T3, free T4, free T3, and TSH were determined in four groups of adult subjects: group A, 27 healthy young volunteers (aged 18-29 yr); group B, 24 carefully selected healthy elderly subjects (aged 70-90 yr); group C, 41 subjects living at a municipal nursing home for the elderly (aged 70-90 yr); and group D, 35 hospitalized patients (aged 70-90 yr). Identical levels of iodothyronines in serum were found in the young and in the elderly healthy subjects. Moderate and severe disease induced alterations in iodothyronine concentrations in serum comparable to those reported earlier. Serum T3 and free T3 levels were low and serum rT3 levels were high in groups C and D subjects; serum free T4 was elevated in group D, while serum T4 was low. Serum TSH was lower in the healthy elderly subjects than in the young subjects. Serum TSH was higher in the elderly sick subjects (groups C and D) than in the healthy subjects (group B). We conclude that advanced age per se is not accompanied by alterations in free or total serum iodothyronine levels.

Adolescent

Clinical investigation of serum deoxyribonuclease: II. Clinical studies of serum deoxyribonuclease activity in pancreatic disease.

Serum Deoxyribonuclease (DNase) of normal persons and of patients with chronic pancreatitis, pancreatic cancer, Diabetes Mellitus, or other malignant diseases was determined with (32P) DNA as substrate. Serum DNase activity was much lower in patients with chronic pancreatitis, pancreatic cancer, or other malignant diseases than in control subjects, and serum DNase activity was almost normal in patients with Diabetes Mellitus. There was no correlation between serum DNase and serum amylase, but there was a good correlation between serum DNase and DNase I output in duodenal juice. There was an inverse correlation between serum DNase and serum RNase. These results imply that in the diagnosis of possible pancreatic disorders serum DNase may be a good indicator and thus may be useful for the detection of malignant diseases.

Amylases

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture.

We studied the effect of fetal calf serum and serum proteins fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296--307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine beta-globulins reduced this transfer, although to a lesser extent than whole serum. alpha-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A gamma-globulin fraction failed to exhibit any effect on the uptake of [14C]phosphatidylcholine by the cells. All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles reincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine ot parenchymal cells. These observation were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of tm proteins. On the other hand, we cannot rule out that plasma membrane modifications are involved in the mechanism of inhibition as well.

Animals