Serum Ultrafiltrate Research Articles

Effects of serum lipid content on the binding of minocycline.

Minocycline was added to normal and hyperlipemic serum samples in concentrations of 1 approximately 10 mcg/ml. These specimens had similar protein contents. Chemically extractable minocycline was quantitated fluorometrically. Hyperlipemic serum (cholesterol 480 mg/100 ml; triglycerides 321 mg/100 ml) yielded an average of 50% less minocycline than did normal serum (cholesterol 170 mg/100 ml; triglycerides 114 mg/100 ml). When ultrafiltrates of serum containing 6, 12 and 20 mcg/ml minocycline were assayed microbiologically, it was evident that variations in serum triglyceride and cholesterol levels did not alter the ratio of bound to free drug. Minocycline appears to be reversibly associated with, and/or soluble in, triglyceride-cholesterol components of serum.

Examination of various cell culture techniques for co-incubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions.

Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecal, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of cell-treponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage of the treponeme was not achieved in vitro.

Characteristics of phosphate transport in isolated proximal tubule.

The characteristics of inorganic phosphate transport in isolated perfused proximal tubules of the rabbit were examined using radioisotopic techniques. When tubules were perfused with an ultrafiltrate of rabbit serum, the mean lumen-to-bath flux of phosphate in the convoluted segment was 6.60 +/- 1.41 (SE) pmol/mm-min with a simultaneous back-to-lumen flux of 0.45 +/- 0.08. In the straight portion of the proximal tubule, the lumen-to-bath flux was significantly lower (P less than 0.01) at 2.22 +/- 0.48 pmol/min-min with a bath-to-lumen flux of 0.31 +/- 0.05. The lumen-to-bath flux was not affected by increases in the intraluminal phosphate concentration from 2.00 +/- 0.19 to 3.12 +/- 0.34 mM or by the isohydric replacement of bicarbonate in the ambient fluids with chloride. However, phosphate absorption was completely inhibited by ouabain 10(-5) M in the bath. These data indicate that phosphate absorption in these segments occurs by a mechanism other than independent diffusion and is saturated at phosphate concentrations characteristic of normal glomerular filtrate. There is no evidence for significant phosphate transport from bath to lumen.

A new device for ultrafiltration The ultrafiltrability of calcium and inorganic phosphate in human serum

The various problems concerning the ultrafiltration of blood serum are out-lined, and a novel ultrafiltration device, which overcomes all these problems is presented. Its main advantages are the following: (1) The deposition of protein on the filter is prevented by producing a flow across the filter surface. (2) The p CO 2 remains constant, since the fluid is not in contact with a gas space. (3) The minimum serum volume is only 0.4 ml (4) Six samples can be processed simultaneously. (5) All commercially available filter types can be used. The machine, tested by measuring the ultrafiltrability of calcium, inorganic phosphate, sodium and chloride in human serum, proved to be a reliable laboratory tool. The precision of the determinations were comparable to respective serum analyses. The ultrafiltrabilities obtained, without correction for water content of serum, were 55.6 ± 2.9% for calcium, 88.3 ± 5.0% for inorganic phosphate, 95 ± 1.7% for sodium, and 103 ± 0.7% for chloride (means ± S.D.).

Protein synthesis and its relation to DNA synthesis in aortic medial explants

Total protein synthesis has been investigated in an in vitro system (swine aortic explants) which has many of the features of the early atherosclerotic lesion. The synthetic activity is measured by the incorporation of [ 14C]leucine into protein the last 2 h of a 9-day culture period. Human sera differ (up to 10-fold) in their capacity to stimulate protein synthesis, and, in addition, cause significantly greater synthesis than normal swine sera when included in the culture medium. The synthetic activity resulting from an ultrafiltrate of serum (mol. wt. 20 000) is less than that resulting from the whole serum of origin, while the recombination of dialyzed serum and dialysate results in enhanced synthesis. There is a high degree of correlation between protein and DNA synthesis measured during this 2-h interval in every experimental situation. Use of metabolic inhibitors shows that DNA synthesis can be almost completely inhibited with no such effect on protein synthesis, but inhibition of protein synthesis causes concomitant depression of DNA synthesis. The explant system may be useful in assessing the importance of the contribution of protein synthesis to the human disease picture.

Inhibition of glyceraldehyde 3-phosphate dehydrogenase by plasma and serum ultrafiltrates due in part to a low-molecular-weight, nonpeptide material

In an attempt to verify the existence in the blood of a diabetogenic peptide (somantin) derived from growth hormone, ultrafiltrates from plasma and serum from normal and diabetic subjects were prepared. The freeze-dried residues of these ultrafiltrates inhibited glyceraldehyde 3-phosphate dehydrogenase as somantin is claimed to do. However, the behavior of the inhibitory material on gel filtration on Sephadex G-10 indicated a molecular weight well below 700, rather than the considerably larger size claimed for somantin. The inhibitory material did not adsorb to Dowex 50W cation exchange resin at pH 2.5, while over 95% of the ninhydrin-positive material was retained. Acid hydrolysis of the inhibitory material did not abolish its activity. Because of the presence of this low-molecular-weight, nonpeptide inhibitory material, inhibition of glyceraldehyde 3-phosphate dehydrogenase by a simple ultrafiltrate of plasma or serum is probably not a definitive measure of somantin.

Binding of 63Ni(II) to Ultrafiltrable Constituents of Rabbit Serum In Vivo and In Vitro

Binding of 63Ni(ll) to ultrafiltrable constituents of rabbit serum was studied (a) after in vitro incubation (2 h, 37 degrees C) of rabbit serum with 63NiCl2 (10-100 mumol/liter), and (b) at intervals (0.25-2 h) after in vivo administration of 63NiCl2(40-160 mumol/kg body wt,i.v.). Serum ultrafiltrates were fractionated by thin-layer chromatography, and the separated compounds made visible by autoradiography and by ninhydrin staining. Severel (congruent to 5) ultrafilitrable 63Ni-complexes were demonstrable as distinct radiodense 63Ni-bands with chromatographic mobilities corresponding to those of ninhydrin-positive bands. Unbound 63Ni(ll) was not detected in serum ultraviltrates in either the in vitro or in vivo experiments. In sera (n equals 10) incubated in vitro with 63Ni(ll) (10 mumol/liter), the mean percentage of ultrafiltrable 63Ni was 36% (range equals 33-38) of total serum 63Ni. In contrast, in sera (n equals 10) obtained 2 h after i.v. injection of 63Ni(ii) (40 mumol/kg), the mean concentration of total serum 63Ni was 10.8 mumol/kg), the mean concentration of total serum 63Ni was 10.8 mumol/liter (ranger equals 6-14), and the mean percentage of ultrafiltrable 63Ni was 15% (range equals 9-21) of total serum 63Ni. The disparity between the percentages of ultrafiltrable 63Ni obtained in vitro and in vivo was obviated when the in vivo experiments were performed in rabbits bilaterally nephrectomized, with ligated common bile ducts. This investigation confirms the existence of several nickel receptors in serum ultrafilitrates and substantiates the role of ultrafiltrable complexes in the excretion of nickel.

Rapid radioimmunoassay of carcinoembryonic antigen (CEA) in ultrafiltered perchloric acid extracts: Results of a clinical survey

Perchloric acid treatment of the carcinoembryonic antigen (CEA) results in subunits of approximate mol.wt 40 000 to 50 000 with different electrophoretic mobilities and antigenic deficiencies as demonstrated by immunoelectrophoresis. An improved radioimmunoassay is described for the quantitation of CEA using rapid ultrafiltration of serum or plasma extracts. The assay was designed to take into account the changes CEA undergoes during perchloric acid extraction. Absorption of antibodies against a crossreacting non-specific antigen from CEA antiserum did not improve the specificity of the radioimmunoassay as was shown by clinical use of the test in almost 1800 patients.

Studies of the activity in chicken serum that stimulates the lymphoid development of the embryonic chicken thymus in organ culture.

Some properties of the thymus stimulating activity in chicken serum (TSAS) which is essential for the lymphoid development of the embryonic chicken thymus in organ culture, have been studied and a partial purification has been performed. TSAS resisted heating for 30 min at 60° C but not higher temperatures. During dialysis and ultrafiltration of serum a loss of TSAS down to a constant level of about 50 per cent of the original activity was obtained. TSAS was sensitive to treatment at pH 5 or lower but resisted alkaline pH up to at least pH 10. When serum was fractionated with ammonium sulphate little TSAS precipitated at 2.43 M ammonium sulphate or lower. At higher concentrations TSAS was found in the precipitates, but even at 4.05 M significant activity remained in the supernatant. TSAS was recovered from a fraction dominated by the serum albumins after gel filtration on Sephadex G‐200 and G‐100. Further fractionation by ion exchange chromatography on DEAE‐Sephadex separated the TSAS from serum albumin. These results suggest that part of the TSAS is associated with one or more specific factors with a molecular weight of approximately 70,000. The effect of the partly purified TSAS on the growth and morphologic development of the embryonic thymus in organ culture was studied.

Alterations of the placental circulation and of there activity of the umbilical artery in experimental hypoxia and asphyxia

In the first series of experiments exteriorization of foetuses was carried out in near-term guinea pigs. The experimental groups of exteriorized foetuses were: 1.Hypoxic /N2 in cap/, 2. asphyctic /temporary cord-clamping in utero/ and 3. controls. The spontaneous closure of the cord vessels, transplacental antipyrine transfer, acid-base balance and survival rate of the foetuses were followed. After induction of spontaneous breathing in the foetuses which remained connected with the placenta, the previousely closed vessels reopened and the placental circulation was restored under the effect of the N2- breathing. In postasphyctie foetuses the placental circulation persisted in spite of the breating of atmospheric air, resulting in a favorable effect on the survival rate. In the second series of experiments the reactivity of isolated human umbilical artery and rabbit central ear artery was studied. The ultrafiltrates of the serum of hypoxic newborns exerted a smaller pressor effect on the human umbilical artery than on the rabbit ear vessel, while the corresponding effect of the normal serum ultrafiltrate was higher on the umbilical artery. In conclusion, a humoral action of the blood of hypoxic-asphyctic newborns is assummed, which tends to preserve the placental circulation.

A colloid osmometer for small fluid samples.

AbstractA membrane colloid osmometer for measurement on samples down to 5 μl has been developed. Pellucid acryl plastic is used for most parts of the osmometer, allowing visual inspection of the reference fluid chamber. The osmometer base is attached directly to a Hewlett‐Packard pressure transducer, replacing the original dome of the transducer, thereby avoiding extra connections and leakage. The exposed membrane area has a diameter of 3 mm, and the membrane may be changed without completely disassembling the osmometer. With Amicon UM‐10 membranes and 0.9 % saline as reference fluid, the equilibration time for measurements on human sera was 2–4 min. Reduction of equilibration time to about 1 min was obtained by “priming” and flushing the membrane with serum ultrafiltrate. Reproducible measurements were obtained on samples down to 4–5 μl.

Binding of 63Ni to rabbit serum α 1-macroglobulin in vivo and in vitro

Nickeloplasmin, the nickel-containing α 1-macroglobulin in rabbit serum, was labelled with 63Ni in vivo and in vitro. Serums were obtained from rabbits at 2 and 8 hours after an i.v. injection of 63NiCl 2. Isolation of serum 63Ni-nickeloplasmin which was labelled in vivo was accomplished by successive fractionations by column chromatography on Sephadex G-200 and by preparative electrophoresis on acrylamide gel. Labelling of nickeloplasmin with 63Ni in vitro was achieved by mixing (a) serum ultrafiltrates from rabbits which had received i.v. 63NiCl 2, with (b) concentrated proteins obtained by ultrafiltration of serums collected from the same rabbits prior to injection of 63NiCl 2. 63Ni-nickeloplasmin preparations which were labelled in vivo or in vitro yielded a single protein band on analytical acrylamide gel electrophoresis and were identified as an α 1-macroglobulin by immunodiffusion and immunoelectrophoresis. Nickeloplasmin was not labelled in vitro after incubation of dialyzed serum proteins with 63NiCl 2 nor after incubation of crude serum macroglobulins with 63NiCl 2. These findings suggest that 63Ni-nickeloplasmin might be a ternary complex of serum α 1-macroglobulin with an ultrafiltrable 63Ni-constituent of serum.

Serum Protein Binding of Erythromycin, Lincomycin, and Clindamycin

Serum protein binding determination for erythromycin, lincomycin, and clindamycin utilizing ultrafiltration of serum containing antibiotic in a concentration of 5 mcg./ml. and tube dilution techniques revealed a high degree of binding: erythromycin base, 73%; erythromycin propionate, 93%; lincomycin, 72%; and clindamycin, 94%.

Sodium chloride and water transport in the medullary thick ascending limb of Henle. Evidence for active chloride transport.

Transport of NaCl and water was examined in the rabbit medullary thick ascending limb of Henle (ALH) by perfusing isolated segments of these nephrons in vitro. Osmotic water permeability was evaluated by perfusing tubules against imposed osmotic gradients. In these experiments the net transport of fluid remained at zero when segments of thick ALH were perfused with isotonic ultrafiltrate in a bath of rabbit serum in which the serum osmolality was increased by the addition of either 239+/-8 mosmol/liter of raffinose or 232+/-17 mosmol of NaCl indicating that the thick ascending limb of Henle is impermeant to osmotic flow of water. When these tubules were perfused at slow rates with isosmolal ultrafiltrate of same rabbit serum as used for the bath, the effluent osmolality was consistently lowered to concentrations less than the perfusate and the bath. That this decrease in collected fluid osmolality represented salt transport was demonstrated in a separate set of experiments in which it was shown that the sodium and chloride concentrations decreased to 0.79+/-0.02 and 0.77+/-0.02 respectively when compared with the perfusion fluid concentrations. In each instance the simultaneously determined transtubular potential difference (PD) revealed the lumen to be positive with the magnitude dependent on the perfusion rate. At flow rates above 2 nl.min(-1), the mean transtubular PD was stable and equal to 6.70+/-0.34 mv. At stop-flow conditions this PD became more positive. Ouabain and cooling reversibly decreased the magnitude of this PD. The transtubular PD remained positive, 3.3+/-0.2 mV, when complete substitution of Na by choline was carried out in both the perfusion fluid and the bathing media. These results are interpreted to indicate that the active transport process is primarily an electrogenic chloride mechanism. The isotopic permeability coefficient for Na was 6.27+/-0.38 x 10(-5) cm.s(-1) indicating that the thick ALH is approximately as permeable to Na as the proximal convoluted tubule. The chloride permeability coefficient for the thick ALH was 1.06+/-0.12 x 10(-5) cm.s(-1) which is significantly less than the chloride permeability of the proximal tubule. These data demonstrate that the medullary thick ascending limb of Henle is water impermeable while having the capacity for active outward solute transport as a consequence of an electrogenic chloride pump. The combination of these characteristics allows this segment to generate a dilute tubular fluid and participate as the principal energy source for the overall operation of the countercurrent multiplication system.

Lipolytic activity of serum and of products of serum ultrafiltration.

Lipolytic activity of serum and of products of serum ultrafiltration.

Penetration of Lincomycin and Clindamycin into Milk in Ewes

SUMMARY The concentrations of lincomycin and clindamycin, both weak organic bases, were determined in serum and milk of lactating ewes after a single and after two intramuscular injections aimed at maintaining relatively constant drug levels in serum. The pharmacokinetic behaviour of the two drugs was very similar. The drugs penetrated within 1 2 h into the milk. Peak milk concentrations of both drugs were higher than peak serum levels and higher levels of the drugs were maintained in milk during at least 11 h after treatment; characteristics which may be utilized in the systemic treatment of mastitis. At constant drug levels in serum the ratio of drug concentration in milk ultrafiltrate to serum ultrafiltrate was close to the calculated ratio derived from the passive diffusion pH-pK partition concept.

Composition of fluid obtained from choroid plexus tissue isolated in a chamber in situ.

1. A method was developed for isolating a segment of the choroid plexus of the lateral ventricle of the cat brain in a chamber in situ.2. A comparison of electrolyte and protein concentrations in serum, ultrafiltrate of serum, cisterna magna fluid, fluid accumulated in the chamber and fluid collected from the choroid plexus by another technique, demonstrates that the chamber fluid is a secretory product of the choroid plexus.3. The rate of fluid formation in the chamber was 0.4 mul. min(-1) mg(-1) of tissue, a value in good agreement with reports in the literature.4. The observation that the concentration of K(+) in choroid plexus fluid was lower than that in the serum ultrafiltrate suggests that K(+) is regulated by an active transport process at the choroid plexus.5. Significant correlation was found between electrolyte values and the protein content of the chamber fluid. This suggests that plasma is the probable source of the protein in the chamber fluid and that K(+) and probably Ca(2+) and Mg(2+) in c.s.f. are subject to active homoeostatic regulation by the choroid plexus.6. The technique described provides a new opportunity to study in detail the functional characteristics of the choroid plexus.

63Ni Complexes in Rabbit Serum and Urine after Injection of 63NiCl2

Abstract Radioactive nickel chloride (63NiCl2) was injected i.v. into rabbits in dosage of 0.24 mg Ni/kg body wt. 63Ni was rapidly cleared from the serum (T1/2 ≃ 8.2 h) during the period from 1 h to 2 days after the injection, but slowly (T1/2 ≃ 95 h) during the period from 4 to 7 days after the injection. During 24 h after injection of 63NiCl2, an average of 90% of serum 63Ni was bound to albumin and 10% was ultrafiltrable. Chromatography on Sephadex G-25 demonstrated the presence of five distinct 63Ni complexes (Fractions I to V) in serum ultrafiltrates. During 24 h after injection of 63NiCl2, an average of 78% of the administered dose of 63Ni was excreted in the urine. Chromatography of the urine on Sephadex G-25 separated three 63Ni complexes, which appeared to have identical chromatographic mobilities as serum Fractions II, III, and V. The chemical identities of the ultrafiltrable 63Ni complexes in serum and urine have not been established, although one (Fraction V) resembles Ni histidine in its chromatographic mobility on Sephadex G-25. This study demonstrates that ultrafiltrable nickel in serum and urine does not exist primarily as free Ni(II), but instead as Ni complexes, and suggests that ultrafiltrable Ni receptors play an important physiological role in nickel homeostasis by serving as diffusible vehicles for the extracellular transport and renal excretion of nickel.

Magnesium deficiency in patients on long-term diuretic therapy for heart failure.

Magnesium levels in serum, erythrocytes, skeletal muscle, and bone were measured in 10 patients with valvular heart disease who had received diuretic therapy for heart failure for an average of 3.3 years. Five patients were found to have diminished values for skeletal muscle, indicating significant magnesium deficit. Values for erythrocytes were low in only two of the five patients, and none had low values for serum ultrafiltrate and bone: Magnesium replacement therapy restored skeletal muscle values to normal. Clinical features consistent with the presence of magnesium deficiency were found in all five magnesium-deficient patients. These features were, with few exceptions, corrected by magnesium replacement. The latter also corrected low skeletal muscle potassium values present in all five patients with low skeletal muscle magnesium, four of whom showed clinical features of digoxin poisoning before magnesium therapy was given. Concomitant secondary aldosteronism, inadequate dietary intake, and digoxin therapy had probably augmented the magnesium loss due to diuretic therapy.

Serum albumin uptake in isolated perfused renal tubules. Quantitative and electron microscope radioautographic studies in three anatomical segments of the rabbit nephron.

Proximal convoluted, proximal straight, and cortical collecting tubular segments isolated from rabbit kidney were perfused with I 125-labeled rabbit serum albumin (RSA-I 125) in ultrafiltrate of serum for up to 3 hr After perfusion, the segments were fixed with glutaraldehyde, embedded in Epon, and either counted with a gamma spectrometer to quantitate protein accumulation or analyzed by electron microscope radioautography to sequentially localize radioactivity Proximal convoluted and proximal straight segments accumulate RSA-I 125 nearly linearly as a function of time whereas cortical collecting segments do not accumulate measurable amounts of protein. The rate of accumulation of RSA-I 125 in the proximal convoluted tubule is 2 6 times as great as that in the proximal straight tubule. Electron microscope radioautography of the isolated proximal tubule demonstrated that RSA-I 125 is taken up via small apical vesicles and tubular invaginations, released into large cytoplasmic vacuoles, and finally concentrated in membrane-bounded structures, some of which are acid phosphatase positive These results show that albumin is absorbed by proximal tubules and may be degraded intracellularly within lysosomes. In addition, less radioactivity was located at all times over the lateral intercellular and basilar labyrinthine spaces, suggesting that labeled albumin and/or its breakdown products may be transported across the peritubular cell membrane.