Human Urine Preparation Research Articles

A fully validated bioanalytical method using an UHPLC-MS/MS system for quantification of DNA and RNA oxidative stress biomarkers.

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Antitumor Activity of Human Urine Extract Cda-2 against Multiple Myeloma Cell Lines Via PI3K/Akt Signaling Pathway in a Caspase-3-Dependent Manner

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment although a number of new agents have recently been shown to be effective. There were still some patients who did not respond to GCs or develop resistance. The aim of this study was to investigate the antitumor activity of human urine extract against MM and Dexamethasone-resistant and sensitive MM cell lines. These cell lines were used to examine the effects of a human urine preparation CDA-2 on the induction of growth arrest and apoptosis. Apoptotic proteins including caspase family, Bcl-2 family were studied. Phosphoinositide 3-kinase (PI3K)/Akt survival signaling pathway was also examined. The caspase-3 inhibitor Z-DEVD-FMK was used to examine the involvement of caspase-3 and PARP. In current study we found that CDA-2 could induce growth arrest and apoptosis of MM cells in vitro. Using MTT assay, we determined the effects of CDA-2 on RPMI8226, U266, MM.1R and MM.1S cells as well as peripheral blood monouclear cells(PBMCs) from three normal volunteers. Fifty percents of growth inhibition (IC50) was measured in these cells treated with CDA-2 at concentrations ranging from 2 to 16 mg/ml for 24 to 72h. CDA-2 exerted substantial growth inhibition in RPMI8226, U266, MM.1R and MM.1S cell lines, while it did not induce cytotoxicity in normal PBMCs. The 24h and 48h IC50 mean values of CDA-2 showed 4.61 and 3.47 mg/ml in RPMI8226 cells(P=0.043<0.05),6.09 and 5.71 mg/ml in U266 cells(P>0.05),5.07and 4.11 mg/ml in MM.1R cells(P=0.002<0.01), 3.26 and 2.13mg/ml in MM.1S cells(P=0.007<0.01). The results indicated that cell viability in the presence of CDA-2 decreased almostly in a dosedependent manner. The inhibitory rates of cell growth were positively correlated with CDA-2 concentrations except U266 cell lines. While in contrasts, CDA-2 did not induce cytotoxicity in PBMCs from three normal volunteers (IC50=103.92 mg/ml, p=0.006<0.01). The mechanism of CDA-2 in MM and Dexmethasone-resistant and sensitive MM cell lines was related to the inhibition of PI3Kp110α expression in protein level, which inactivated the phosphorylation of Akt involving dephosphorylation of Bad protein, downregulation of Bcl-xl protein, and triggered the activation of caspase cascades. We contrasted the grey scale of the PI3Kp110αprotein level after treated with the different concentrations of CDA-2. The grey scale values of 8226 cells and MM.1R cells after treated with 4mg/ml CDA-2 were 3.71 A2.11 respectively, while the grey scale values of the untreated control cells of 8226 and MM.1R were 6.92 A7.46 respectively (P=0.001<0.01). This phenomenon could be inhibited by the caspase-3 inhibitor Z-DEVD-FMK. In addition, CDA-2 could also downregulate the level of DNA maintenance methylation enzyme, DNMT-1 in MM and Dexamethasone-sensitive and resistant MM cell lines. Therefore, our results demonstrate the presence of active components in the human urine extract that can induce growth arrest and apoptosis of Multiple Myeloma and Dexamethasone-resistant and sensitive Multiple Myeloma cell lines and may involve the PI3K/Akt signaling pathway in a caspase-3 dependent manner, associated with downregulation the level of the DNA maintenance methylation enzyme. This may provide new insights for the treatment of MM, especially the drug-resistant Multiple Myeloma.

Human urine extract CDA-2 induces apoptosis of myelodysplastic syndrome-derived MUTZ-1 cells through the PI3K/Akt signaling pathway in a caspase-3-dependent manner1

The aim of this study was to investigate the antitumoral activity of human urine extract against myelodysplastic syndrome (MDS)-derived MUTZ-1 cells in vitro and in vivo. The MDS-refractory anemia with excess of blasts (RAEB)-derived MUTZ-1 cell line was used to examine the effects of a human urine preparation, CDA-2, on the induction of growth arrest and apoptosis. Apoptotic proteins, including caspase family, Bcl-2 family, the inhibitor of apoptosis protein (IAP) family, and the FLICE-like inhibitory protein (FLIP), as well as cell cycle-associated proteins were studied. The phosphoinositide 3 kinase (PI3K)/Akt survival signaling pathway and the NF-kappaB pathway were also examined. The caspase-3 inhibitor Z-DEVD-fmk was used to examine the involvement of caspase-3 and poly (ADP-ribose) polymerase (PARP). PI3K inhibitor LY294002 was used to examine the involvement of the PI3K/Akt signaling pathway in this apoptosis-inducing effect. MUTZ-1 cell xenografted serious combined immunodeficiency disease mice were used for the in vivo study. We found that CDA-2 could induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo. The main mechanisms were related to the inhibition of PI3Kp110alpha expression at the transcriptional level, which inactivated the phosphorylation of Akt involving the prevention NF-kappaB phosphorylation and nuclear translocation, the downregulation of the IAP family and FLIPL protein, and the dephosphorylation of the Bad protein, which then triggered the activation of the caspase cascades. This phenomenon could be inhibited by the PI3K inhibitor LY294002 and caspase-3 inhibitor Z-DEVD-fmk. Our results demonstrate the presence of active components in the human urine extract that can induce the growth arrest and apoptosis of MDS-RAEB-derived MUTZ-1 cells and may involve the PI3K/Akt signaling pathway in a caspase-3-dependent manner. This may provide new insights for the treatment of high-risk MDS.

Differentiation of Pheochromocytoma PC12 Cells Induced by Human Urine Extract and the Involvement of the Extracellular Signal–Regulated Kinase Signaling Pathway

The aim of this study was to investigate the effects of a human urine preparation on the differentiation of tumor cells. The pheochromocytoma PC12 cells were used to examine the effects of a human urine preparation, CDA-2 on the induction of differentiation markers, neurofilaments, and compared with that induced by nerve growth factor (NGF). The MAPK/ERK kinase (MEK) inhibitor U0126 was used to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathway in this differentiation inducing effect. We find that CDA-2 could induce differentiation of pheochromocytoma PC12 cells, as evidenced by the markedly increased expression of neurofilaments to a level comparable to those induced by NGF. This phenomenon was accompanied by the phosphorylation of extracellular-signal-regulated kinase (ERK) and could be inhibited by the MEK inhibitor, U0126. Our results demonstrate the presence of active components in the human urine extract that can induce differentiation of PC12 cells and may involve the ERK signaling pathway. This may provide new insights for seeking novel differentiation agents and offer hope for cancer patients.

Rapid preparation of human urine and plasma samples for analysis of F 2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F 2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F 2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F 2-isoprostane showed recovery of 55–65% and high linearity for concentration 0–1.0 ng/ml for urine (CV=4.08%, r 2=0.990) and 0–0.5 ng/ml for plasma (CV=4.07%, r 2=0.998). Fasting for 6 h significantly increased plasma F 2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.

Effect of CDA-II on cell viability, lipid peroxidation, glutathione concentration and its related enzyme activities in primary rat hepatocytes.

This study investigated the effects of various concentrations and incubation time intervals of CDA-II (cell differentiation agent: a preparation of human urine) on cell viability, lipid peroxidation and glutathione and its related enzyme activities in rat hepatocytes. Based on the results of lactate dehydrogenase leakage and microscopic examination, treatment with CDA-II significantly elevated the viability of hepatocytes. The level of lipid peroxidation of cells treated with 2.5 or 5 mg/ml CDA-II was lower than the control after 4, 8 and 24 hours of incubation. Intracellular glutathione (GSH) content of cells treated with 0.25-5 mg/ml CDA-II was significantly higher than controls after 8 and 24 hours of incubation. These phenomena are beneficial to the antioxidant capabilities of hepatocytes. Furthermore, treatment of hepatocytes with CDA-II has no effect on the activities of GSH peroxidase, GSH reductase and GSH S-transferase. In conclusion, adequate concentration of CDA-II could enhance the viability and antioxidative capability of hepatocytes.

Effect of CDA-II, urinary preparation, on lipofuscin, lipid peroxidation and antioxidant systems in young and middle-aged rat brain.

The levels of lipofuscin and lipid peroxidation reflect the degree of free radical-induced oxidative damage in the brain. We examined the effects of CDA-II, a preparation of human urine, on lipofuscin and lipid peroxidation in the brain of young (3.5 months) and middle-aged rats (17 months). The rats were given CDA-II orally at dosages of 0.3 or 1.0 g/kg daily for 8 weeks. CDA-II significantly suppressed the contents of lipofuscin and lipid peroxidation in both young and middle-aged rats. CDA-II also elevated the activity of superoxide dismutase, and the amounts of glutathione and ascorbic acid in the middle-aged rats, but not in the young ones. Our results suggest that the protection against oxidative damage by CDA-II in the young rat brain may be due to its scavenging activity against free radicals. In the middle-aged rats, in addition to the scavenging activity, the levels of endogenous antioxidants were also enhanced by the CDA-II treatment.

Scavenging of reactive oxygen species by a urinary preparation.

In the present paper, the antioxidant properties of a preparation of human urine (PHU) were evaluated by studying the ability of this drug to react with relevant biological oxidants such as superoxide anion radical (O2*-) and hydroxyl radical (OH*). In addition, its effect on lipid peroxidation was investigated in vitro and ex vivo. PHU is not a good scavenger of O2*-. However, it reacts rapidly with OH radicals with a second-order rate constant of 2.8 x 10(9)/M/sec. The studies on rat brain homogenates showed that PHU had an inhibitory effect, which was dependent on its concentration and the magnitude of lipid peroxidation. Ex vivo studies also showed that oral administration of PHU increased the antioxidant capacity of plasma from rats. The ability of PHU to scavenge free radicals suggests that this drug may be potentially useful in counteracting free radical-mediated diseases.

Effect of a Urinary Preparation on Liver Injury by Short-term Carbon Tetrachloride Treatment in Rats

The hepatoprotective effect of a preparation of human urine (PHU) was assessed against short-term carbon tetrachloride (CCl4) treatment in rats. Significant prevention of liver injury by PHU was found after CCl4 treatment, judging by the changes of serum biochemical parameters, and hepatic protein and triglyceride contents. The increased liver lipid peroxidation, and decreased liver vitamin C concentrations observed after CCl4 treatment were significantly prevented by PHU administration. The increase in liver glutathione (GSH) contents observed after CCl4 treatment was further increased by PHU treatment. Liver catalase activity decreased after CCl4 treatment, while liver superoxide dismutase and GSH-peroxidase activities did not change. PHU administration further inhibited the decrease in liver catalase activity after CCl4 treatment. These results indicate that PHU administration can prevent liver injury induced by CCl4 in rats by inhibiting enhanced lipid peroxidation and by improving disrupted active oxygen metabolism in the injured liver.

Ameliorative effect of an urinary preparation on acetaminophen and D-galactosamine induced hepatotoxicity in rats.

The effect of oral administration of a preparation of human urine (PHU) on acute liver injury was examined in rats intoxicated with acetaminophen and D-galactosamine. The results indicated that PHU protected the liver from acetaminophen and D-galactosamine-induced injury as judged by morphological and biochemical observation. An increase in lipid peroxide concentrations and decrease in protein concentrations occurred in the liver by D-galactosamine injection, PHU administration significantly prevented these changes.

Effect of pretreatment of rats with an urinary preparation on liver injuries induced by carbon tetrachloride and alpha-naphthylisothiocyanate.

The effect of oral administration of a preparation of human urine (PHU) on the progression of acute liver injury was examined in rats intoxicated with carbon tetrachloride (CCl4) and alpha-naphthylisothiocyanate (ANIT) PHU protected the liver from CCl4-induced injury as judged by morphological and biochemical observations. In contrast, PHU aggravated ANIT-induced injury as judged also by morphological and biochemical observation. PHU prevented the increase in hepatic glutathione (GSH) and lipid peroxidation induced by CCl4. But PHU enhanced the increase in hepatic GSH caused by ANIT. These results indicate that the effect of PHU on hepatic GSH concentrations is through an indirect pathway. Clinical application of PHU on hepatitis should be explored further.

Affinity chromatography of urokinase on an agarose derivative coupled with pyroglutamyl-lysyl-leucyl-argininal.

Pyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the epsilon-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2 X 10(-4)M.

Haematological effects in mice of partially purified colony stimulating factor (CSF) prepared from human urine.

Summary. Single injections of partially purified human urine preparations containing 10 000–20 000 units of colony stimulating factor (CSF) produced a monocytosis and polymorph leucocytosis in neonatal C57BL mice which was maximal at 48 hr. Adult C57BL mice developed a monocytosis, and although no polymorph leucocytosis developed, labelling studies suggested an increased rate of granulopoiesis. In adult mice the injected CSF preparations did not alter total bone‐marrow cellularity but increased the percentage of primitive granulocytes and caused an increase in the number of in vitro colony‐forming cells and granulocytic cluster‐forming cells. The preparations also increased the number of bone‐marrow cells able to initiate colony and cluster formation immediately after culture of the bone‐marrow cells in agar. The results suggest that CSF in vivo may stimulate both granulopoiesis and monocyte formation in an action similar to its action in agar cultures of bone‐marrow cells.

Serum Half Life in Mice of Colony Stimulating Factor Prepared from Human Urine

Summary. Partially purified preparations of the colony stimulating factor (CSF) from human urine were given as a single intravenous injection to adult C57BL mice. Assays for human CSF in the sera of these mice were performed in cultures of C57BL bone marrow cells using a discriminatory rabbit anti‐human CSF antibody preparation, incapable of blocking the action of mouse serum CSF. The half life of injected CSF varied from 1.7 to 3.7 hr (mean 2.9 hr). The injection of these human urine preparations caused a temporary elevation of mouse CSF levels in the serum of recipient mice, which prolonged the effective period of elevated CSF levels in the serum following injection.

Hemoglobin Switching in Nonanemic Sheep. III. Evidence for Presumptive Identity between the A→ C Factor and Erythropoietin

Abstract The degree of A → C hemoglobin switching following administration of a human urine erythropoietin preparation to a non-anemic sheep was compared, in the same animal, with the switching obtained with administration of anemic sheep plasma containing known amounts of erythropoietin. A strong correlation emerges between the dosage of erythropoietin given and the degree of A → C switching observed. This correlation between erythropoietin dose and A → C response is maintained despite the use of materials that originated in different species (man and sheep), were assayed for erythropoietin in yet another species (mouse), and had grossly different specific activities (units/mg.) of erythropoietin, so that the specific activity of the human urine preparation was approximately 400 to 1600 times that of anemic sheep plasma. We believe these observations provide substantial evidence for a provisional hypothesis of identity between erythropoietin and the A → C switching factor.