Comparative metabolomics analysis on hematopoietic functions of herb pair Gui-Xiong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach.

The compatibility of Angelicae Sinensis Radix (Danggui, DG) and Chuanxiong Rhizoma (Chuanxiong, CX), a famous herb pair Gui-Xiong (GX), can produce synergistic and complementary hematopoiesis. In present study, global metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) combined with pattern recognition method was performed to discover the underlying hematopoietic regulation mechanisms of DG, CX and GX on hemolytic and aplastic anemia rats (HAA) induced by acetyl phenylhydrazine (APH) and cyclophosphamide (CP). Thirteen endogenous metabolites contributing to the separation of model group and control group were tentatively identified. The levels of LPCs including lysoPC (18:0), lysoPC (20:4), lysoPC (16:0) and lysoPC (18:2), sphinganine, nicotinic acid, thiamine pyrophosphate, phytosphingosine, and glycerophosphocholine increased significantly (p<0.05) in HAA, while the levels of oleic acid, 8,11,14-eicosatrienoic acid, ceramides (d18:1/14:0), and 17a-hydroxypregnenolone decreased significantly (p<0.05) in comparison with control rats. Those endogenous metabolites were chiefly involved in thiamine metabolism and sphingolipid metabolism. The metabolic deviations could be regulated closer to normal level after DG, CX and GX intervention. In term of hematopoietic function, GX was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of metabolomic strategy, reflecting the synergic action between DG and CX. The relative distance calculation was firstly used in metabolomics for semi-quantization.

Effects of various antireabsorptive treatments on bone mineral density in hypogonadal young women after allogeneic stem cell transplantation.

Ovarian failure after allogeneic stem cell transplant (allo-SCT) is an important risk factor for development of osteoporosis. We investigated the effects of various antiresorptive treatments in long-term surviving females with ovarian failure after allo-SCT. A total of 60 women with osteoporosis or osteopenia were divided randomly into four groups of 15 women each. Group 1 was treated with calcium and vitamin D alone, group 2 received the same treatment in combination with hormone replacement therapy (HRT), group 3 received risedronate (35 mg weekly, orally for 1 year) and group 4 zoledronic acid (3 monthly doses of 4 mg (intravenous)). All groups were similar for age, body mass index, underlying disease and time elapsed from transplant. Lumbar and femoral bone mineral density (BMD) were measured at baseline and after 12 months, together with serum osteocalcin and urinary hydroxyproline. At 12 months, a significant decrease in lumbar and femoral BMD was observed in group 1 and a milder decrease in group 2. Risedronate treatment increased significantly lumbar BMD and prevented bone loss at the femoral neck. Zoledronic acid increased significantly both lumbar and femoral BMD. In groups 3 and 4 the hydroxyproline excretion was significantly reduced, while osteocalcin mildly increased only in group 4. In conclusion, bisphosphonate administration is useful to prevent and treat bone demineralization in young adult women after allo-SCT.

Hemangiopoietin, a novel human growth factor for the primitive cells of both hematopoietic and endothelial cell lineages.

The cells of hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblast. However, the existence of a growth factor acting relatively specifically on hemangioblasts remains unclear. Here we report the identification of hemangiopoietin (HAPO), a novel growth factor acting on both hematopoietic and endothelial cell lineages. In vitro in the human system, recombinant human HAPO (rhHAPO) significantly stimulated the proliferation and hematopoietic and/or endothelial differentiation of human bone marrow mononuclear cells and of purified CD34+, CD133+, kinase domain receptor-positive (KDR+), or CD34+/KDR+ cell populations. In the murine system, rhHAPO stimulated the proliferation of long-term culture-initiating cells (LTC-ICs) as well as CD34+ and stem cell antigen-1 (Sca-1+) cell subsets. In vivo, subcutaneous injection of rhHAPO into normal mice resulted in a significant increase in bone marrow hematopoietic cells. Furthermore, irradiated mice injected with rhHAPO had an enhanced survival rate and accelerated hematopoiesis. Our data suggest that HAPO is a novel growth factor acting on the primitive cells of both hematopoietic and endothelial cell lineages and that HAPO may have a clinical potential in the treatment of various cytopenias and radiation injury and in the expansion of hematopoietic and endothelial stem/progenitor cells.

Comparison of oral iron chelator L1 and desferrioxamine in iron-loaded patients.

The efficacy of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) was compared with that of subcutaneous desferrioxamine in 26 patients with transfusional iron overload. Immediately after red-cell transfusion, 20 patients were randomised to receive either desferrioxamine (50 mg/kg daily as a 12 h subcutaneous infusion), or L1 (50 mg/kg daily by mouth). Patients were evaluated during treatment with the other drug after transfusion the next month. Mean (SD) daily urinary iron excretion was lower during L1 than during desferrioxamine (12.3 [6.7] vs 18.2 [15.3] mg/day). In 5 patients the dose of L1 was raised from 50 to 75 mg/kg daily; mean urinary iron excretion rose from 13.8 (7.0) mg/day to 26.7 (17.8) mg/day, comparable with that during desferrioxamine (24.9 [24.3] mg/day). Faecal iron excretion rose slightly over baseline in 6 patients studied during L1 administration (from 8.5 [0.9] mg/day to 12.2 [0.9] mg/day). Pharmacokinetic studies showed an elimination half-life for L1 of 117-237 min. Studies in dogs and in volunteers showed no absorption of the L1-iron complex, excluding a contribution of absorption of intraluminal complexes of L1 and food iron to urinary iron excretion. Further animal toxicity testing is needed before L1 can be studied in a broader group of patients.

Purification of human urine colony-stimulating factor by affinity chromatography.

Antisera from rabbits immunized with murine macrophage colony-stimulating factor (CSF-1) were evaluated for cross-reactivity with human urine CSF-1. One cross-reactive antiserum was used to purify CSF from human urine. The IgG fractions from normal rabbit serum and the anti-CSF serum were bound to cyanogen bromide-activated sepharose. Ten-liter pools of human urine were concentrated by ultrafiltration and applied sequentially to the normal IgG and antiserum columns. Cross-reactive proteins were removed by the IgG column, whereas CSF was bound by the anti-CSF column. After extensive rinsing of the antibody column, the CSF was eluted with 4 M sodium thiocyanate. This fraction contained four to five contaminating proteins as judged by migration in sodium dodecyl sulfate-acrylamide gel. In a further purification step, the CSF was retained selectively by concanavalin A sepharose and eluted with alpha-methylglucoside. This purified CSF had a specific activity of 0.8-2.3 x 10(7) U/mg protein. A single major contaminant was removed by reversed phase high performance liquid chromatography. Final specific activity of the purified CSF ranged from 2.5 to 4.4 x 10(7) U/mg protein. Each 10-liter pool of urine yielded 18-20 micrograms of pure material with a 15%-25% recovery. This technique is more rapid and provides a higher yield of pure human CSF-1 than the more tedious multi-step procedures that have been described previously.

Production of monoclonal antibodies against human erythropoietin and their use in the purification of human urinary erythropoietin.

Several murine monoclonal antibodies (MAbs) specific for human erythropoietin (HuEpo) were produced by hybridomas obtained from the fusion of murine myeloma cells, P3X63-Ag.8-653, with the splenocytes of mice immunized with recombinant human Epo (rHuEpo). Based on epitope analysis by a competitive binding assay, these MAbs could be classified into at least three groups: (1) 1E10, (2) 1H7, (3) 2D6, 3D6 and 3D8. In a sandwich enzyme-linked immunosorbent assay (ELISA), using these MAbs as the solid-phase antibodies, MAb-bound HuEpo was detected with rabbit anti-HuEpo sera. Some combinations of two different classes of MAbs, such as 1H7 and 3D8, were found to capture much more HuEpo than each MAb used individually. Urinary HuEpo (U-HuEpo) was highly purified from the urine of patients with severe aplastic anemia with about 50% final recovery using an immunoaffinity column on which a mixture of 1H7 and 3D8 was immobilized. The purified U-HuEpo had a specific activity of 77,340 U/mg in a radioimmunoassay (RIA) and of 76,673 U/mg using an in vivo bioassay.

Effects of an aplastic anemia urinary extract on mouse erythroid progenitor cells in vivo.

We investigated the in vivo effects of a crude extract from the urine of aplastic anemia patients (AA urinary extract) on erythroid precursor cells in the femoral bone marrow and spleens of normal adult mice. A single intraperitoneal injection of AA urinary extract induced a significant increase in the number of splenic erythroid burst-forming units (BFU-e) and erythroid colony-forming units (CFU-e) within 24 h after injection. We then injected pure recombinant erythropoietin (Epo) equivalent to the amount present in the urinary extract. This addition increased the number of splenic CFU-e by almost the same degree as the amount induced by the AA urinary extract 24 h after injection, but failed to elicit any change in the number of splenic BFU-e. In other studies, mice were injected with the same amount of lipopolysaccharide (LPS) and/or pure Epo as that present in the AA urinary extract. Experiments with Limulus amebocyte lysate-adsorbed (endotoxin-depleted) or nonadsorbed (endotoxin-containing) AA urinary extracts showed that endotoxin contamination interfered with the increase in numbers of marrow CFU-e and enhanced the increase in splenic CFU-e numbers induced by pure Epo or Epo activity in the AA urinary extract. The number of splenic BFU-e, however, was not affected by administration of LPS and/or Epo or by adsorbed endotoxin. These data suggest that AA urinary extract contains a stimulating activity for mouse splenic BFU-e, and that this activity is not attributable to the Epo activity or endotoxin contamination within the urinary extract.

Granulopoietic effects of colony-stimulating factor obtained from urine of patients with aplastic anemia on normal and cyclophosphamide-treated mice.

Colony-stimulating factor (CSF) was partially purified from urine of patients with aplastic anemia using DEAE-cellulose and concanavalin A-Sepharose. This partially purified CSF caused significant neutrophilia in the peripheral blood of normal mice by (a) single or continual intraperitoneal injection(s) in vivo, and also revealed a specific activity of 1.4 x 10(3) U/absorbance unit (AU) at 280 nm in vitro, with less than 1 ng/AU endotoxin. In addition, this CSF induced faster recoveries of neutrophils in the peripheral blood and progenitor spleen cells of cyclophosphamide (CY)-treated mice. These findings suggest that the CSF used in this study accelerated the differentiation of the granulocytic cells and the proliferation of granulocyte colony-forming units in the spleen. These effects contributed to a rapid recovery from neutropenia in mice treated with CY.

Effects of urinary extracts from patients with idiopathic thrombocytopenic purpura or aplastic anemia on rodent platelet production and megakaryocytopoiesis.

Urinary extracts from idiopathic thrombocytopenic purpura (ITP) patients, aplastic anemia (AA) patients and normal subjects were investigated for their effects on in vivo platelet production, and both in vitro and in vivo megakaryocytopoiesis in rodents. Daily intraperitoneal injection of 1.2 absorbance units (AU, A278) of urinary protein for three consecutive days induced statistically significant increases in rat blood platelet numbers. This increase was observed for 1 of 4 ITP urinary extracts and for all 3 AA urinary extracts, and occurred 24 h after the final injection. In vitro levels of megakaryocyte colony-stimulating factor (Meg-CSF) in ITP urinary extracts were similar to those of normal urinary extracts, and were in dramatic contrast to the markedly elevated levels of Meg-CSF in extracts from AA urine. A single intraperitoneal injection of 0.5 AU of AA urinary protein induced a significant increase in spleen-derived megakaryocyte colony-forming cells (CFU-meg) 48 h past injection. In the group injected with ITP urinary extract, CFU-meg levels remained within normal limits. These results provide evidence that urinary extracts of ITP patients do not contain increased levels of Meg-CSF and a factor which directly stimulates in vivo CFU-meg production, and that the decrease in circulating platelet numbers that is characteristic of ITP patients is not a primary in vivo determinant in the elaboration of these factors.

Neuraminidase and hematopoietic factors from human urine.

Human urinary neuraminidase, an enzyme that releases sialic acid from hematopoietic factors found in urinary preparations, was partially characterized, and a method was developed to derive these hematopoietic factors free of enzyme activity. Neuraminidase in urinary preparations from healthy humans and aplastic anemic (AA) patients had optimal activity at pH 5.3 and hydrolyzed both alpha 2----3 and alpha 2----6 type ketosidic linkages of N-acetyl-neuramin lactose and alpha 1-acid glycoprotein. When subjected to Sephacryl S-300 gel filtration, urinary neuraminidase showed a single peak of activity with an apparent molecular weight of 380,000 daltons, even under denaturing conditions (6 M guanidine hydrochloride). Furthermore, among a variety of compounds tested, no potent inhibitor of the enzyme was found. Heat treatment of AA urinary preparations eliminated about 80% of neuraminidase activity, while successive two-step ethanol precipitation eliminated residual enzyme. Erythropoietin, megakaryocyte colony-stimulating factor (CSF) and granulocyte/macrophage phage CSF activities were retained after these treatments.

The humoral regulation of megakaryocytopoiesis and platelet production in vivo.

We examined the effects of the urinary extracts from aplastic anemia (AA) patients, idiopathic thrombocytopenic purpura (ITP) patients, and normal subjects on murine megakaryocyte/platelet production in vivo and in vitro. In the first study, single doses of AA urinary protein (65%-90% ethanol precipitate) were individually injected intraperitoneally into rats and mice. Blood platelet counts in rats increased significantly 24 hours after the injection. Total megakaryocyte colony-forming units (CFU-Meg) in mouse spleens increased by 24 hours postinjection, peaked at 48 hours and returned to normal levels at 96 hours. Changes in the number of megakaryocyte colonies showed similar patterns of increasing, peaking and returning to normal levels postinjection. In the second study, we compared the effects of some urinary extracts on murine megakaryocyte/platelet production. These observations provided the evidence that AA urinary extracts contain a factor that directly stimulates megakaryocyte progenitor cell proliferation in mouse spleen in vivo as well as the release of platelets from megakaryocytes, and ITP urinary extracts do not contain increased levels of Meg-CSF and/or some other factor that directly stimulates CFU-Meg in vivo, and the decreased blood platelet mass that is clinically characteristic of ITP is not a primary in vivo determinant of the elaboration of these factors.

Isolation of human erythropoietin with monoclonal antibodies.

Human erythropoietin was isolated from urine of aplastic anemic patients in a high yield with a simple purification procedure using an immunoadsorbent column of monoclonal antibodies and a Sephadex G-100 column. About 6 mg of erythropoietin was isolated from 700 liters of urine and the specific activity was estimated to be 81,600 units/mg of protein with an in vivo 59Fe incorporation assay method, using starved rats. Activity measurement of the extracts from sliced gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blotting technique revealed heterogeneity of the isolated erythropoietin, which is probably caused by variable amounts of carbohydrates attached to the polypeptide chain. Thirty amino acids in the NH2-terminal portion of the isolated hormone were sequenced.

In vivo stimulation of murine granulopoiesis by human urinary extract from patients with aplastic anemia.

Significant in vivo stimulation of granulopoiesis was induced in mice by the administration of an extract from the urine of patients with aplastic anemia (AA). Sialic acid has been identified as an important molecular component for the in vivo biological activity of this granulopoietic factor, "granulopoietin," which is distinct and different from endotoxin. Urine from patients with AA was successively fractionated by Sephadex G-50 and DEAE-cellulose chromatography. The resultant extract, which we refer to as AA urinary extract, contained approximately equal to 44 international units of erythropoietin per A unit of protein and induced 15,000 colonies of granulocyte/macrophage precursor cells (granulocyte/macrophage colony-forming units, CFU-gm) per A unit of protein with mouse bone marrow. Eight daily intraperitoneal injections of this extract in mice induced a 6.2-fold increase in peripheral blood granulocytes and a 14.6-fold increase of splenic CFU-gm, with concomitant increases in the proliferation rates of CFU-gm in both bone marrow and spleen. Pretreatment of the AA urinary extract with sialidase significantly diminished these granulopoietic effects in vivo (P less than 0.001). In contrast, both extracts (i.e., native AA and sialidase-treated AA urinary extracts) revealed high granulocyte/macrophage colony-stimulating factor activity in vitro when clonal assays were performed with mouse bone marrow. Increased in vivo and in vitro granulopoietic activities were found in the concanavalin A "break-through" fraction, indicating that these activities were due to protein(s) that did not bind to the lectin. These results reveal that this urinary extract from patients with AA is capable of inducing significant granulopoiesis in mice and that sialic acid is an important component in the maintenance of this granulopoietic effect in vivo but not in vitro.

Partial purification and biological properties of thrombopoietin extracted from the urine of aplastic anemia patients.

Thrombopoietic (Tpo) and megakaryocyte-colony stimulating (Meg-CSF) activities were found in urinary extracts from patients with aplastic anemia. Preparative biochemical extractions were accomplished using Sephadex G-50 and DEAE-cellulose column chromatography. The biological activities of these extracts were assessed using not only an in vivo assay but were also examined in vitro employing the clonal development of megakaryocyte colonies. Both in vivo, as well as in vitro, biological activities were detected in the batch fraction which was stepwise eluted from DEAE-cellulose between 0.022 M NaCl in 0.016 M NaH2PO4 and 0.15 M NaCl in 0.05 M Na2HPO4 as a single fraction. When 0.4 mg of this fraction was injected daily into rats, a marked thrombopoiesis ensued producing an increase of 40% over initial platelet counts by 3 days after administration. This was followed by a decrease in platelets to a subnormal range by 21 days after the injection. Hemoglobin concentration gradually increased from 5% above initial value by day 7 to 20% above initial value by day 21. The effect of neuraminidase (NAse) on the properties of this extract was also examined. NAse-treated extracts, similar to the native extracts described above retained Tpo activity. Changes in megakaryocyte numbers in the spleen and bone marrow of rats were assayed with both the NAse-treated extract as well as with the native extract. A remarkable increase in megakaryocyte numbers, threefold above the normal count, was found in the spleens of rats given the native extract preparation; by contrast, however, no change was observed in splenic megakaryocyte numbers in rats given the NAse-treated extract. On the other hand, NAse-treated extract retained its ability to stimulate bone marrow megakaryocyte proliferation in the same rat. The urinary extract also revealed in vitro Meg-CSF activity with a specific activity of 31, 750 CFU-Meg colonies/mg of protein.

Thrombopoiesis and megakaryocyte colony stimulating factor in the urine of patients with aplastic anaemia.

The urinary extracts from patients with aplastic anaemia and from healthy donors were investigated in vivo and in vitro for their ability to stimulate megakaryopoiesis and platelet production. There was a significantly higher concentration of thrombopoiesis stimulating factor (TSF) and megakaryocyte colony stimulating factor (MEG-CSF) in the urine from patients with aplastic anaemia than in that from healthy donors. Neuraminidase treatment did not affect the thrombopoietic activity of TSF, whereas coexisting erythropoietin (EPO) in the extract lost in its activity in vivo. These findings suggest that TSF and/or MEG-CSF seems to be different from EPO and that the urine from aplastic anaemia patients would be a good source of TSF and MEG-CSF for purification and characterization.