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At least 19 recordsLinked to original sources

PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗

PubMed1979

Immunotitration of alkaline phosphatase isozymes in normal and pathological urine.

Isoenzyme patterns of alkaline phosphatase are determined quantitatively in extracts of human kidney as well as in human urine by means of immunotitration technique. Both media contain two types of AP isoenzymes: liver and intestinal like AP. Intestinal AP is located as a minor component of total AP activity (1-4%) in particle-free fraction of the kidney. Urinary AP activity is found after high speed centrifugation in supernatant (100,000 Xg) as well as in the 100,000 Xg sediment and can only be made soluble from the latter by n-butanol treatment. Intestinal AP in urine is concentrated in the supernatant while in sediment the isoenzyme pattern resembles to that of kidney. Urine of normal persons contains most of AP activity in the sediment and consists mainly of liver type AP. Urinary AP of patients with renal diseases or after application of cytotoxins contains little sedimentable activity, mainly intestinal AP.

Alkaline Phosphatase↗

PubMed1977

[Collagenase activity in an animal and a human carcinoma (author's transl)].

High amounts of collagenase were found in chemically-induced carcinomas of the mouse skin and in carcinomas of the human urinary bladder. Part of the total enzyme activity was detected in the supernatant after sedimentation of the tumour homogenate at 6000 x g and dialysis for 48 hours. The remainder was extracted from the pellet by the use of 5 M urea. Though the localization of enzyme production and regulation of enzyme activity is still unclear, the collagenases may be implicated in the breakdown of collagen structures during tumour invasion.

Animals↗

PubMed1977

Adsorption of mercury to urinary cells or particles.

Urine of a young healthy man was added with varying amounts of mercuric chloride, incubated for 10 to 375 min, and separated into supernatant solution and precipitate by centrifugation at 105,000 X g for 60 min. Percentage of mercury amount in precipitate increased with the increase of mercury amount added. But this adsorption of mercury to urinary cells or particles could not completely account for the amount of mercury in human urine reported (Suzuki and Shishido 1975). The hypothesis that desquamation of tubular cells is a route of urinary mercury excretion in man is still sustained.

Adsorption↗

PubMed1975

The metabolism of 3-chloro,- 3-bromo- and 3-iodoprpan-1,2-diol in rats and mice.

1. The metabolism of the 3-halopropan-1,2-diols (alpha-halohydrins) has been investigated in rats and mice. Apart from 3-chloropropan-1,2-diol (I), of which some 10% is excreted unchanged by both species, the compounds are completely degraded following intraperitoneal administration. 2. The alpha-halohydrins are detoxicated by conjugation with glutathione and produce two urinary metabolites, isolated and identified as S-(2,3-dihydroxypropyl)cysteine (VII) and the corresponding mercapturic acid N-acetyl-S-(2,3-dihydroxypropyl)cysteine (VIII). 3. When incubated with rat liver supernatant, the compounds do not conjugate with glutathione and their general chemical reactivity suggests that they react via a common intermediate proposed to be glycidol (2,3-epoxypropanol, IV). As the epoxide produces the same urinary metabolites as the alpha-halo-hydrins, and conjugates with glutathione either with or without liver supernatant to form the primary metabolite S-(2,3-dihydroxypropyl)glutathione (VI), glycidol is also proposed to be the reactive intermediate in vivo. 4. The role of epoxides in intermediary metabolism is discussed.

1-Propanol↗

PubMed1975

Desquamation of renal epithelial cells as a route of mercury excretion in man: a preliminary study.

Urine of mercury-exposed workers in a chlorine manufacturing factory and of staffs of our laboratory was separated in the supernatant solution and the precipitate by a centrifugation of 105,000 Xg for 60 min. The ratio of the amount of inorganic mercury in the precipitate to that in the supernatant solution ranged from 0.23 to 4.84, and was dependent upon the level of urinary mercury. The role of the desquamation of tubular cells is suggested as a possible route of urinary excretion of mercury.

Adult↗

PubMed1976

Determination of oxalic acid in urine by atomic absorption spectrophotometry.

A method is described for determination of oxalic acid in urine using atomic absorption spectrophotometry. The concentration of urinary oxalic acid is calculated by using the difference between two determinations of calcium: first, the excess of calcium in the supernatant after precipitation as calcium oxalate at pH 5, and second, the total calcium determined at pH less than 1 (endogenous and added). Analytical parameters (pH of precipitation, temperature, calcium and oxalate added, precipitation time, interfering substances) were studied with the aid of [14C]oxalic acid. A constant recovery of 95% of total oxalic acid allows the use of a correction factor. The accuracy and reproducibility of the method make it useful for routine determination of urinary oxalic acid.

Evaluation Studies as Topic↗

PubMed2026

CASTOR1 Regulates Humoral Immune Responses and Contributes to the Pathogenesis of Systemic Lupus Erythematosus.

OBJECTIVE: CASTOR1 senses arginine and regulates mammalian target of rapamycin complex 1 (mTORC1), a central metabolic signaling molecule. This study aimed to elucidate the roles of CASTOR1 in humoral immune responses. METHODS: We analyzed human B cell transcriptomes from healthy controls and patients with systemic lupus erythematosus (SLE) via correlation analysis and gene set variation analysis using our database, Immune Cell Gene Expression Atlas from the University of Tokyo. Castor1-deficient and B cell-specific Castor1-deficient mice were used for analyses of serum immunoglobulins and autoantibodies, urinary proteins, renal pathology, gene expression, and flow cytometry in spleen and bone marrow cells. The culture supernatant of splenic B cells was used for immunoglobulin (Ig) analysis. RESULTS: Transcriptomic analysis of bulk RNA sequencing data from various B cell subsets in patients with SLE (n = 136; n = 129 included in the primary analysis) revealed a correlation between CASTOR1 expression and disease activity, with CASTOR1 expression in plasmablasts inversely correlated with Systemic Lupus Erythematosus Disease Activity Index 2000 (r = -0.32, P = 0.00031). Castor1-deficient mice exhibited increased plasma cell populations in the spleen and bone marrow, elevated serum IgG levels, production of anti-double-stranded DNA antibodies, and glomerulonephritis with IgG deposits, reflecting SLE-like autoimmunity. Moreover, B cell-specific Castor1-deficient mice showed increased plasma cell counts, elevated serum IgG levels, and glomerulonephritis, indicating that Castor1 might regulate systemic humoral immunity via a B cell-intrinsic mechanism. CONCLUSION: CASTOR1 plays a regulatory role in humoral immunity and may contribute to the pathogenesis of autoimmune diseases such as SLE, representing a potential therapeutic target.

Journal Article↗

PubMed1979

Determination of urinary 4-pyridoxic acid using high performance liquid chromatography.

A high performance liquid chromatographic method is presented for the determination of the vitamin B6 metabolite, 4-pyridoxic acid, in urine. Urine samples are treated with trichloroacetic acid to precipitate protein. An aliquot of the supernatant is chromatographed using 0.033 M phosphate buffer containing 5% (v/v) methanol (pH 2.2), a fluorometric detector and a commercial reverse phase octadecylsilica column. The high precision of the method and the absence of interfering compounds have been demonstrated. This method provides a rapid, sensitive, and quantitative technique for the measurement of urinary 4-pyridoxic acid for use in metabolic and nutritional studies.

Chromatography, High Pressure Liquid↗

PubMed1979

Studies on rat renal cortical cell kallikrein. II. Identification of kallikrein as an ecto-enzyme.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha''-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.

Animals↗

PubMed1977

Metabolic disposition of cytembena in rats and dogs.

1. The metabolic disposition of cytembena (sodium cis-3-p-methoxybenzoyl-3-bromoacrylate)labelled at both carbonyl carbon atoms was studied in rats and dogs. 2. Rats excreted over 70% and dogs excreted over 50% of the radioactive dose in 24 h. Most of the radioactivity was found in urine. Kidney retained the highest level of radioactivity after 24 h. Renal cortex was responsible for the observed high retention. Both species exhibited similar tissue distribution and urinary metabolic patterns. 3. Cytembena was metabolized by demethylation catalysed by microsomes and by debromination and double bond saturation catalysed by the 100,000 g supernatant fraction in the presence of glutathione and NADPH. Tentative structures of the metabolites identified by mass spectrometry and the probable metabolic pathway of cytembena are presented,

Acrylates↗

PubMed1992

T-cell alterations in immunoglobulin A nephropathy.

In the present paper are reported alterations of the cellular immunity found in 24 patients with immunoglobulin A nephropathy (IgAN). CD4+ and CD25+ cells were increased in patients in comparison with controls. The mean of interleukin-2 (IL-2) and interleukin-4 (IL-4) levels in sera were similar in patients and controls, but the levels of IL-2 in supernatants of stimulated peripheral blood mononuclear cells from patients were higher than those of controls. There were no correlations between renal function, serum IgA levels, urinary findings, cellular subsets, and IL-2 or IL-4 sera levels. These immunological data were also unrelated to the mode of clinical presentation. The results suggest a pivotal role of IL-2 in cellular immune response with regard to T-cell activation in patients with IgAN.

Adult↗

PubMed1977

Effects of maleic acid administration on urinary excretion of DCEC, and on tissue protein, in the rat.

Administration of maleic acid to rats caused increased urinary excretion of S-(1,2-dicarboxyethyl)cysteine [DCEC] and increased binding of maleic acid to tissue proteins. The trichloroacetic acid precipitate from a combined fraction of supernatant and microsome gave the maximum amount of DCEC after hydrolysis. It would appear that aminoaciduria produced by maleate was partially due to increased destruction of some succinylated tissue proteins.

Amino Acids, Dicarboxylic↗

PubMed1977

Oversaturation of urine with sulphadiazine during treatment with a small therapeutic dose.

The urinary crystallization of N1-pyrimid-2-yl-sulfanilamide (sulphadiazine, Sulfolex) during treatment with a small therapeutic dose (600 mg/day p.o.) for 6 days was evaluated in seven voluntary female subjects. In 5/7 and 3/7, respectively, of the urine supernatants obtained from samples collected 6 and 24 h after the last dose of sulphadiazine, the sulphonamide concentration exceeded the experimental solubility of the drug in 37 degrees C urine at the same pH. However, only in 3 sediments of the 6-h urine samples could a few sulphadiazine crystals be found. The findings suggest that the urine often can be oversaturated in respect to sulphadiazine, without any marked crystallization.

Adult↗

PubMed1977

Immunocytochemical localization of lysozymes in respiratory and other tissues.

Immunostaining paraffin sections of appropriately fixed tissues with an antiserum to human urinary lysozyme as the primary step in an immunoglobulin-peroxidase bridge method has localized lysozyme in previously recognized sites such as Paneth cells, renal tubules, and lymph node macrophages in several species. In addition, lysozyme was demonstrated in the ciliary layer of the trachea, and type II pneumocytes, as well as cells of presumed mucoid nature in laryngotracheal glands. Large stellate cells in follicle centers in the lymph nodes and spleen and in the medulla of the thymus evidenced strong lysozyme reactivity. Granular pneumocytes disclosed immunoreactivity for lysozyme also at the ultrastructural level. Lysoplate assay demonstrated lysozyme in abundance in both the cellular pellet and acellular supernatant of rat alveolar wash fluid and in rat lung after repeated washing of alveoli. Hamster lung differed from the others in failing to immunostain for lysozyme and affording no evidence for content of lysozyme as determined by lysoplate assay. Sites stained with antiserum to human urinary lysozyme failed to stain with antiserum to egg white lysozyme. However, the pyloric glands, Golgi elements in intestinal epithelium, the surface of the colon, and the proximal straight renal tubule of the mouse stained exclusively with the antiserum to hen egg white lysozyme. Many sites staining with antiserum to urinary lysozyme in respiratory, renal, and lymphoid tissue lacked reactivity in control sections exposed to this antiserum after it was absorbed with purified urinary lysozyme. However, mucous acini in submandibular glands, although failing to stain with other control procedures, retained towared the absorbed antiserum, possibly through reacting with an antibody other than that for human urinary lysozyme. A number of cell types containing proteinaceous cytoplasmic granules stained in control sections exposed to normal serum in place of antilysozyme serum in the immunoglobulin-peroxidase bridge procedure and, thus, possessed selective, but nonimmunospecific affinity for immunoglobulin. Cell types that stained with antiserum to hen egg white lysozyme lost affinity for the antiserum after its absorption with egg white lysozyme but retained the affinity after absorption with urinary lysozyme.

Animals↗

PubMed1978

Urinary glycopeptides and glycosaminoglycans in children and adults as affected by age.

The glycopeptide and glycosaminoglycan excretion was studied in the urine of normal human subjects from newborns to 68 years of age. The glycopeptide and glycosaminoglycan preparations were made by digesting the urine with peoteolytic enzymes followed by removal of enzyme and undigested protein and finally freeze-drying of the supernatant. The contents of hexose, sialic acid and uronic acid were determined on these preparations. Because of the striking differences in body weight all values were expressed in terms of milligrams per 24 hours per kilogram of body weight. When corrected for body weight, all of the carbohydrate levels were significantly higher in infants and young children under 5 years of age than in older children and adults. No other significant changes with age were found after the age of 7 years. There were no significant differences between the sexes at any age.

Adolescent↗

PubMed1977

The influence of fasting on the disposition of warfarin in rats.

A study was undertaken to investigate the influence of fasting on the disposition of warfarin in rats. Fasting consisted of withholding solid food, but not water, immediately following warfarin (3 or 10 mg/kg s.c.) administration until sacrifice at different time intervals (3, 6, 12, 24 and 30 hours). Control animals were fed ad libitum. Total and unbound warfarin concentrations were measured in plasma and liver supernatant as well as unchanged warfarin and its metabolites in urine. The disposition of unbound warfarin was found to be markedly affected by fasting, especially at the 3 mg/kg dose: the disappearance rate of unbound warfarin from plasma was accelerated in fasted animals in contrast to that of total warfarin. In addition, unbound warfarin was cleared from plasma at a more rapid rate than total warfarin in both control and fasted animals. At the 10 mg/kg dose, the disposition of total and unbound warfarin was little affected by fasting. The concentration of unbound warfarin in the liver supernatant of fasted rats given warfarin, 3 mg/kg, was significantly increased at 6 and 24 hours of fasting. Plasma free fatty acids were significantly elevated starting at 6 hours, but no such difference was noticed with liver homogenate, except at 24 hours. The 24-hour urinary excretion of unchanged warfarin was higher in fasted rats, but fasting failed to produce any change in the excretion of warfarin metabolites. The results of the present investigation indicate that short periods of fasting influence the disposition of unbound warfarin without apparently modifying its biotransformation and further show the importance of plasma protein binding on the pharmacokinetics of warfarin.

Animals↗

PubMed1979

The effect of sulphur on 75Se absorption and retention in sheep.

Four Border-Leicester X Merino wethers were used in a 4 X 4 Latin square experiment to study the effects of dietary sulphur on selenium absorption and retention. The basal diet contained 0.05% S and sodium sulphate was added to give additional treatment levels of 0.11, 0.17 and 0.24% total sulphur. Sodium selenate was added to all diets to bring the dietary selenium level to a constant 0.25 mg/kg. One hundred muCi 75Se as sodium selenate (specific activity 50 muCi/mg Se) was administered to the rumen per fistulam after a 10-day period of adjustment on each diet. Radioactivity in blood, urine, faeces and rumen digesta was measured at intervals over the succeeding 7 days. Twenty percent of the total activity in the rumen fluid was in the TCA supernatant fraction after 3 hours, and this proportion tended to increase slightly as sulphur intake increased. Fecal excretion of selenium accounted for between 44 and 51% of the dose after 7 days, the high levels being associated with increasing sulphur intake. However, these differences were not significant. Urinary excretion of selenium accounted for between 12% (0.05% S) and 22% (0.24% S) of the dose after 7 days, with treatment differences being significant. Levels of radioactivity in blood were significantly higher in sheep fed the 0.05% S diet compared with those fed the higher levels. The results show that sulphur affects apparent selenium excretion and suggest that the metabolism of these two elements is intimately related.

Absorption↗