Liter Of Serum Research Articles

In vivo studies on anti hyperglycemic activity of sericin using rat model

Silk protein, sericin was isolated from raw silk fabrication in reeling industry and subsequent process of silk throwing. The majority of it is thrown in the waste water from silk manufacturing. Sericin was used in antibacterial, antioxidant, wound healing, moisturising, and antiaging medications and cosmetics. Limited reports on the use of sericin in the treatment of diabetes prompted the current study on the use of sericin in the control of diabetes in rats. Thus, the present study was aimed to investigate the antihyperglycemic activity of sericin protein using rat model. The results indicated that sericin treated groups showed a noteworthy augment in body weight than the standard drug (Metformin) treatment with diabetic control. Sericin @ 400mg/kg showed 152.6 mg/dl and 141.2 mg/dl on 7th and 21st day of treatment. Similarly, Sericin @ 800mg/kg showed 126.0 mg/dl and 114.0 mg/dl on 7th and 21st day of treatment when compared to control (206.0 mg/dl and 203.6 mg/dl). SGOT of 68.4 and 50.0 units per litre of serum was observed in sericin @ 400 mg/kg and 800mg/kg treated rats when compared to control 138.6 units. SGPT 0f 26.0 and 21.6 units per litre of serum was observed in sericin @ 400 mg/kg and 800mg/kg treated rats when compared to control (75.0 units). Triglycerides of 96.4 and 81. 4 units were observed in sericin @ 400 mg/kg and 800mg/kg treated rats when compared to control (152.8 units).

Antithymocyte Globulin at Clinically Relevant Concentration Kills Leukemic Blasts

Background/Rationale: Success of allogeneic hematopoietic cell transplantation (HCT) is limited due to graft-versus-host disease (GvHD) and leukemia relapse. Contrary to small molecule immunosuppressive drugs like cyclosporine or methotrexate, rabbit antithymocyte globulin (ATG) used for GvHD prophylaxis with myeloablative HCT conditioning does not increase relapse. The mechanism of the anti-GvHD effect of ATG has been studied extensively, however, the reason for ATG not increasing relapse is not known. We hypothesized that ATG has an anti-leukemic activity. Here we investigated the anti-leukemic activity of ATG in vitro at concentrations achieved in vivo with our standard ATG dose of 4.5 mg/kg (median 10 mg per liter of serum; range, 1-25). We focused on complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC).Methods: Blood or bone marrow from patients newly diagnosed with acute myeloid leukemia (AML, n=25) or acute lymphoid leukemia (ALL, n=4), obtained before induction chemotherapy, was used as the source of leukemic cells. We measured CDC and CIC of ATG, specifically against leukemic blasts at clinically achieved concentrations (1-25 mg/L) and an achievable concentration (50 mg/L). The leukemic blasts were identified as CD45dim/neg and side scatter low (SSclow) cells in case of AML-M0, M1 or M2 (according to British-American-French classification) or ALL. In case of AML-M4 or M5, we gated on CD45dim/neg and SSclow/intermediate cells. These cells could contain both leukemic and normal blasts and some post-blast (maturing/mature) cells. We minimized the fraction of normal blasts and post-blast cells included by introducing a "dump channel". The dump channel consisted of antibodies against antigens expressed by cells of lineages other than the leukemia lineage (except for antigens aberrantly expressed by the leukemic cells) and by post-blast cells. For most AML patients, the dump channel consisted of CD10, CD19, CD41a, CD235a, CD14 and CD16. For most ALL patients, the dump channel consisted of CD117, CD33, CD41a, CD235a, CD14 and CD16. Thus, leukemic blasts were defined as mononuclear cells that were CD45dim/neg and SSclow (or SSclow/intermediate for M4/M5) and did not express dump channel antigens. CDC was induced by incubation of the patient specimen with human serum (source of complement). CIC was induced by incubation of the patient specimen with heat-inactivated serum (no complement). For CDC dead cells were identified as 7-amino-actinomycin D positive (7AAD+). For CIC dead/dying cells were identified as Annexin V positive (Annexin V+).Results: ATG induced death of leukemic blasts both via CDC and CIC. Median 0.3%, 3.0%, 12.6% and 41.7% blasts were killed with 1, 10, 25 and 50 mg/L ATG, respectively, via CDC. Median 2.1%, 8.0%, 11.4% and 12.6% blasts were killed after 4 hour incubation with 1, 10, 25 and 50 mg/L ATG, respectively, via CIC. For both CDC and CIC, the percents were significantly higher than background (p<0.05). There was a high variability in the sensitivity of the leukemic blasts to ATG. The adjusted (background subtracted) percent of blasts killed ranged from 2.6% to 93.8% via CDC (Figure 1) and from 1.5% to 69.9% via CIC (Figure 2). When we arbitrarily defined a patient with resistant leukemic blasts as <10% (adjusted) leukemic blasts dead after exposure to 50 mg/L ATG, there were 3/28 patients with resistant leukemic blasts in case of CDC (Figure 1) and 9/23 patients in case of CIC (Figure 2).Conclusion: ATG at clinically relevant concentrations kills primary leukemic blasts in vitro. Some acute leukemias are highly sensitive and others relatively resistant to ATG. Whether this applies to in vivo needs to be determined. If yes, and given that the killing of leukemic cells is ATG concentration-dependent, conditioning regimens with high dose ATG could be developed for patients with leukemia sensitive to ATG to maximize the concurrent anti-GvHD and anti-relapse effects. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Serum and tissue angiotensin converting enzyme in patients with lichen planus

Serum and tissue angiotensin-converting enzyme (ACE) was measured in 20 patients with lichen planus before and after therapy, and in 20 healthy individuals. Serum and tissue ACE activity was determined by spectrophotometric method using hippuryl-l-histidyl-l-leucine as a substrate. The enzyme activity is expressed in the following units: 1 U corresponds to 1 nmol of hippuric acid released by hydrolysis of hippuryl-l-histidyl-l-leucine per minute and one liter of serum or 50 mg tissue. Before therapy, serum ACE activity was significantly increased in patients with lichen planus (35.9 +/- 2.33 U/L) in comparison to healthy individuals (28.16 +/- 1.7 U/L). Tissue ACE activity was increased in patients with lichen planus (2.24 +/- 0.41 U/50 mg) in comparison to healthy individuals (1.86 +/- 0.16 U/50 mg), but the difference was not significant. After therapy, serum and tissue ACE activity decreased and no significant difference in ACE activity was found. The determination of serum ACE activity may be a good non-specific parameter for the assessment of therapeutic effects.

Indications that paraoxonase-1 contributes to plasma high density lipoprotein levels in familial hypercholesterolemia

HDL-associated paraoxonase type 1 (PON1) can protect LDL and HDL against oxidative modification in vitro and therefore may protect against cardiovascular disease. We investigated the effects of PON1 levels, activity, and genetic variation on high density lipoprotein-cholesterol (HDL-C) levels, circulating oxidized LDL (OxLDL), subclinical inflammation [high-sensitive C-reactive protein (Hs-CRP)], and carotid atherosclerosis. PON1 genotypes (L55M, Q192R, -107C/T, -162A/G, -824G/A, and -907G/C) were determined in 302 patients with familial hypercholesterolemia. PON1 activity was monitored by the hydrolysis rate of paraoxon, diazoxon, and phenyl acetate. PON1 levels, OxLDL, and Hs-CRP were determined using an immunoassay. The genetic variants of PON1 that were associated with high levels and activity of the enzyme were associated with higher HDL-C levels (P values for trend: 0.008, 0.020, 0.042, and 0.037 for L55M, Q192R, -107C/T, and -907G/C, respectively). In addition to the PON1 genotype, there was also a positive correlation between PON1 levels and activity and HDL-C (PON1 levels: r = 0.37, P < 0.001; paraoxonase activity: r = 0.23, P = 0.01; diazoxonase activity: r = 0.29, P < 0.001; arylesterase activity: r = 0.19, P = 0.03). Our observations support the hypothesis that both PON1 levels and activity preserve HDL-C in plasma.

Pentachlorophenol Exposure in Women with Gynecological and Endocrine Dysfunction

Exposure to wood preservatives containing pentachlorophenol (PCP) was detected in 65 women who consulted the Endocrinological Department of the University Hospital of Obstetrics and Gynecology, Heidelberg, Germany, because of gynecological problems. Blood PCP levels ranged from 20.7 to 133 μg per liter of serum. One hundred and six women with similar clinical conditions, corresponding age and body weight, no PCP exposure in history, and PCP levels below 20 μg per liter of serum served as control group. Significant associations were found between serum PCP concentrations, age, and different parameters of the endocrine system. PCP may act centrally on a hypothalamic or suprahypothalamic level which may result in mild ovarian and adrenal insufficiency. PCP may, therefore, play a role in the increasing infertility problem.

The Angiotensin-Converting Enzyme (ACE) Gene Insertion/Deletion Dimorphism Tracks with Higher Serum ACE Activities in Both Younger and Older Subjects

The absence of a 287 base pair alu sequence in the ACE gene (D allele) is associated with higher ACE levels than its presence (I allele) in adults. We carried out a case-control study of the ACE*I/D dimorphism in relation to circulating ACE activities to evaluate associations between the two variables in adults, compared to younger (18 years or less) individuals. Genotypes of the ACE*I/D dimorphism were determined on DNA samples from a population of 164 random (unrelated) Emirate nationals, composed of two groups: 112 subjects above 18 years of age (range=20-77 years), and 52 subjects of 18 years or less (range=1-18), and analyzed for putative associations with serum ACE activities. ACE*I/D genotypes of the 164 individuals were determined by assays based on polymerase chain reaction. ACE activities were determined on serum samples of these subjects by colorimetric assays. The D allele was associated with increased ACE values in both adult and younger individuals. Mean ACE activity levels associated with II, ID and DD genotypes, however, were 42%-61% higher in the 18 years and under group of subjects. The ACE*I/D marker accounted for 28% of the variance of the phenomenon determining ACE levels in adults, and for 30% among youngsters. The ACE*I/D dimorphism correlated strongly with circulating ACE activities in both adult and young Emirati subjects, and the corresponding mean ACE activities were significantly higher among the youngsters.

Measurement of Procainamide and N-Acetylprocainamide in Serum by High-Performance Liquid Chromatography

We measured procainamide (I) and its metabolite, N-acetylprocainamide (II), in human serum samples by solvent extraction, high-performance liquid chromatography on a reverse phase column, and detection at 280 nm, with use of external standards. The method requires 0.2 ml of serum and is sensitive to 0.3 mg of I and 0.6 mg of II per liter of serum, with intra-assay standard deviations of 0.22 and 0.24 mg/liter, respectively, at 5 mg/liter (N=10) and inter-assay standard deviations of 0.63 and 0.81 mg/liter, respectively, at 7.5 mg/liter (CV 8.4 and 10.5%, respectively, n = 20). Concentrations measured by high-performance liquid chromatography and by an established fluorescence technique correlated well (r = 0.98 for I and 0.97 for II). No interfering substances were found in 20 randomly selected sera from patients receiving a large number of other drugs. Of the pure drug substances tested only sulfathiazole interfered with the assay of II. The method is therefore suitable for routinely monitoring these compounds in serum in a clinical laboratory. The high concentrations of the metabolite in a significant number of patients demonstrate the need to consider it as well as the parent drug as guides in optimizing dosage regiments for I.

Elimination of Human Chorionic Gonadotropin in Sandwich Enzyme Immunoassay for Human Thyroid-Stimulating Hormone in Serum

Abstract A method to eliminate human chorionic gonadotropin (hCG) in the sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. hTSH in serum containing hCG was reacted with dinitrophenyl monoclonal mouse anti-hTSH β-subunit IgG1, and the complex formed between the dinitrophenyl IgG1 and hTSH was trapped onto affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. hCG in the test serum was largely eliminated by washing the polystyrene balls. Subsequently, the complex on the polystyrene balls was reacted with affinity-purified rabbit anti-hCG Fab′-peroxidase conjugate followed by washing. The complex of the dinitrophenyl IgG1, hTSH and the conjugate was eluted with dinitrophenyl-L-lysine from the polystyrene balls, to which hCG had been nonspecifically adsorbed, and was trapped onto clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. Peroxidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls in the absence and presence of hTSH was not significantly affected by the presence of up to 75,000 IU of hCG per liter of serum. As a result, serum hTSH could be sensitively measured with little interference by hCG.

Assay of dipeptidyl peptidase IV in serum by fluorometry of 4-methoxy-2-naphthylamine.

A new fluorometric assay for determining dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) was developed. The synthetic substrate glycyl-L-proline-4-methoxy-2-naphthylamide (20 mmol/L), Tris buffer (50 mmol/L, pH 8.3), and serum (20 microL) are mixed and incubated. The reaction is stopped with citrate (100 mmol/L, pH 4.0) and the released 4-methoxy-2-naphthylamine is measured fluorometrically. The mean value of DPP IV activity in serum for 64 healthy subjects was 58 (SD 16) mumol of 4-methoxy-2-naphthylamine released per liter of serum per minute. The proposed procedure is sensitive, rapid, and accurate and can easily be automated.

A new enzymatic assay of chloride in serum.

This new method for enzymatic assay of serum chloride is based on determination of chloride-dependent alpha-amylase (EC 3.2.1.1) activity. We evaluated the ion specificity and practicability of the method for routine use with the Hitachi 705. The analytical range of the method extends from 40 to 160 mmol of chloride ion per liter of serum. The reaction rate for samples containing 100 mmol of chloride per liter was 0.17 A/min. CVs within-run and between-run were less than 1.0%. Correlation with results of a coulometric titration method was good. The specificity for bromide is 75% of that for chloride; for the other anions, it is 0%. This enzymatic method is generally applicable to wide variety of automated chemistry analyzers.

Two-monoclonal-antibody "sandwich"-type assay of human lutropin, with no cross reaction with choriogonadotropin.

We have developed a sensitive, specific, noncompetitive sandwich-type assay for human lutropin (hLH). Two monoclonal antibodies are used, and there is no cross reaction with human choriogonadotropin (hCG) or human follitropin (hFSH), and little or none with human thyrotropin (hTSH). There also is no reaction with the free beta chains of hLH and hCG. The detection limit is less than 0.5 int. units of hLH per liter of serum, and the dose-response curve is linear between 0 and 10 int. units/L. The intra-assay CV averaged 5.4% at low doses of hLH; the interassay CV averaged 12.5%.

Direct chemiluminescence immunoassay for estradiol in serum.

In this simple, reliable, fast solid-phase chemiluminescence immunoassay for directly measuring (i.e., without prior extraction) estradiol-17 beta in serum, a monoclonal antibody is used that binds estradiol with high affinity (Ka = 10(10) L/mol), and does not bind other steroids tested, the highest cross reactivity observed being 0.1% for estradiol-17 alpha. In this system the monoclonal antibody is bound to the wells of microtiter plates via a second antibody directed against the monoclonal antibody. Fifty microliters of serum and estradiol-displacing agents are added, followed by 100 pg of estradiol-isoluminol conjugate, and the label is measured by luminometry after the binding reaction. The sensitivity of the assay is 180 pmol per liter of serum, and the effective working range at less than or equal to 10% CV is 270 to 6700 pmol/L. Analytical recovery of added estradiol averaged 99.7% (SD 6.5%). Within- and between-assay CVs ranged between 5 and 12.7%. Thirty-five unknown serum samples can be assayed within 4 h. Results correlated well with those obtained with a direct RIA: r = 0.94 (n = 149). This assay opens new perspectives for chemiluminescence immunoassays.

Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody.

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.

Determination of serum ferritin by a one-step immunoenzymoassay, and comparison of four liver-ferritin standards.

We describe a one step "sandwich"-type immunoenzymoassay for ferritin in human serum. The solid-phase consists of glutaraldehyde-treated polypropylene tubes coated with rabbit antibody to human ferritin. Liver ferritin is the standard. Peroxidase-conjugated antiserum to ferritin and a sensitive chromogen, o-phenylenediamine, are used. The assay requires 90 min. The standard curve is linear up to 400 micrograms of ferritin per liter of serum. Within- and between-run CVs are less than 6% for low, high, and medium concentrations and are about 13.0% at the decision level for iron deficiency. Results by a two-step "sandwich" procedure (New England Immunology Associates kit) correlated well, r = 0.98. We assessed four liver ferritin standards from different manufacturers with the described method. The mean absorbance for the 40 micrograms/L ferritin standard was 1.5 for that from Diagnostics Biochem and National Institute for Biological Standards and Controls, 1.0 for that from Dako, and 0.4 for that from Sigma. Consequently, to standardize results, all liver ferritin standards should be calibrated vs the National Institute for Biological Standards and Controls reference standard.

Standardized automated assay for functional alpha 1-antitrypsin.

We describe an enzymic assay for the functional activity of alpha 1-antitrypsin, in which tosyl-Gly-Pro-Lys-p-nitroanilide acetate (Chromozym PL) is the trypsin substrate. For the indicating reaction, the concentration of substrate is about double the Km of its reaction with trypsin. This assay is an improvement over reactions involving N-alpha-benzoyl-DL-arginine p-nitroanilide as substrate, the solubility of which in water is less than the Km of its reaction with trypsin. The assay is standardized in terms of active sites of trypsin inhibited per liter of serum, with p-nitrophenyl-p'-guanidinobenzoate as the active site titrant. The results agreed well with those by an immunonephelometric assay for alpha 1-antitrypsin (r = 0.953). The within-run and run-to-run precision was approximately 3%. The results of the assay varied linearly with alpha 1-antitrypsin concentration from 0 to 60 mumol/L of serum. The minimum sample size was 50 microL, and analysis of 24 samples took less than 20 min.

Enzyme immunoassay for pregnancy-associated plasma protein-A.

This enzyme immunoassay procedure for pregnancy-associated plasma protein type A involves a "sandwich"-type system in microtitration plates. One can detect 0.27 mg of the protein per liter of serum, with a between-batch CV of 10.2%, and the antiserum used does not cross react with any of the other placentally derived proteins. A reference interval for the last trimester of pregnancy is presented. The procedure described is suitable for studying the behavior of this protein during pregnancy.

Radioimmunoassay of aspartate aminotransferase isoenzymes in human serum.

We describe the development of a sensitive, specific radioimmunoassay for the cytoplasmic and mitochondrial isoenzymes of human aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1). Isoenzymes from human heart tissue were purified to homogeneity and used to raise high-titer antisera in rabbits. We partly purified the antisera by selective column chromatography. The Bolton-Hunter reagent was used to radioiodinate the isoenzymes. The assay requires 100 microL of serum, includes a solid-phase second-antibody separation, and can be completed in less than 3 h. There was no cross reactivity between the two isoenzymes. As little as 5 micrograms (50 pmol) of each aspartate aminotransferase can be measured per liter of serum.

The effect of chronic bile loss on the thyroid gland

The morphofunctional state of the parenchyma and the microcirculatory bed of the thyroid gland in chronic bile loss were studied. The studies were carried out on 25 cats of both sexes, in which external drainage of the common bile duct was performed from 5 to 40 days. After 7-10 days, pronounced hypertrophy and hyperplasia of the follicular epithelium were detected in the thyroid gland. Hyperplasia was extrafollicular in nature with the formation of numerous microfollicles. There was a statistically significant increase in the height of the follicular epithelium cells and a decrease in the average diameter of the follicles. On the 10th day of the experiment, the level of protein-bound iodine in serum was 0.290.03 /o 131I per 1 liter of serum (Functional research methods in endocrinology. Kiev, Zdorov'ya, 1981), in the control - 0.34 0.04%. Accumulation of 131I reached its maximum level after 6-10 hours.

Human prostatic acid phosphatase: purification, characterization, and optimization of conditions for radioimmunoassay

Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48 000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with α-naphthyl phosphate at 30°C and para-nitrophenyl phosphate at 37°C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 μg of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9–7.7% with serum at 1.3 to 5.6 μg PAP/l.

Automated assay for alpha 1-antitrypsin with N-alpha-benzoyl-DL-arginine-p-nitroanilide as trypsin substrate and standardized with p-nitrophenyl-p'-guanidinobenzoate as titrant for trypsin active sites.

alpha 1-Antitrypsin is the most abundant of several serum protease inhibitors. Its deficiency is associated with an increased incidence of emphysema in adults, jaundice in newborns, and childhood cirrhosis. We describe an automated functional assay for the Instrumentation Laboratory's Multistat III Microcentrifugal Analyzer with N-alpha-benzoyl-DL-arginine-p-nitroanilide as trypsin substrate. The assay is standardized in terms of moles of trypsin active sites inhibited per liter of serum, by use of a chromogenic titrant for trypsin active sites, p-nitrophenyl-p'-guanidinobenzoate. The method is rapid, precise, and independent of trypsin supplier, and results correlate well with those by a manual chromogenic and a nephelometric assay.