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.

Megakaryocyte colony-stimulating factor (Meg-CSF) is a unique cytokine specific for the megakaryocyte lineage.

The regulation of megakaryocytopoiesis and platelet production has not yet been clearly elucidated. Several cytokines have been shown to be capable of producing megakaryocyte colonies from bone marrow [i.e. Interleukin (IL)-3, granulocyte-macrophage (GM)-colony-stimulating factor (CSF), erythropoietin (Epo)]. In addition, other activities have been reported to stimulate megakaryocyte precursors, yet a megakaryocyte-CSF (Meg-CSF) has not been purified to homogeneity and IL-3, GM-CSF and/or Epo often contaminate purification attempts which could account for the activities. A Meg-CSF has been isolated from the urine of patients with aplastic anaemia and purified by sequential ultrafiltration, cation exchange, G-50 chromatography, preparative PAGE, chromatofocusing and cation exchange HPLC. The activity of this material is 2-4 x 10(4) CFU-Meg/mg as measured in a murine marrow, serum-containing assay. This activity also stimulates CFU-Meg in the absence of adherent accessory cells and in serum-free cultures, indicative of the direct stimulation on CFU-Meg. Immunoassays, colony forming assays, and proliferation assays demonstrate that purified Meg-CSF has no GM-CSF, IL-3, M-CSF, G-CSF or IL-1 alpha, -3, -6, -9 and -11. In confirmation of these results, neutralizing antibody to IL-6 also did not abrogate Meg-CSF activity. Therefore the previously-reported megakaryocyte colony-stimulating activity in purified aplastic anaemia patient urine is due to a unique cytokine: Meg-CSF.

3-Methylglutaconic aciduria associated with Pearson syndrome and respiratory chain defects.

3-Methylglutaconic aciduria was detected in four patients with Pearson syndrome, a multitissue disorder with hematologic abnormalities, lactic acidosis resulting from defective oxidative phosphorylation, and deletions in the mitochondrial genome. 3-Methylglutaconic acid may be an additional useful marker for Pearson syndrome and may be a more specific marker than other organic acids identified in this disorder.

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.

Partial purification and characterization of human megakaryocyte colony-stimulating factor (Meg-CSF).

Megakaryocyte colony-stimulating factor (Meg-CSF) in urinary extracts from patients with aplastic anemia was partially characterized and purified. Using Meg-CSF-enriched fractions, we established that the moiety has the following characteristics: 1) portions of the molecules having Meg-CSF activity have sialic acid, probably with a biantennary structure, and beta-galactose residues as the terminal and penultimate sugars; 2) disulfide residues are an essential chemical group of the molecule and are located on its surface; and 3) Meg-CSF activity is stable in n-propanol, but not in acetonitrile with trifluoroacetic acid. Partial purification of Meg-CSF by a four-step procedure of ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-sepharose chromatography, and high-resolution hydroxyapatite chromatography, yielded a concentrate with a 430- to 630-fold increase in specific activity. The partially purified Meg-CSF fractions stimulated both human and murine megakaryocytopoiesis in vitro (CFU-meg). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreduced conditions, Meg-CSF activity was recovered in the 29-34 kDa molecular weight fractions. We have also shown that Meg-CSF, purified from the urine of aplastic anemia patients, stimulated murine megakaryocytopoiesis and platelet production in vivo. Final purification of human urinary Meg-CSF is currently in progress.

Human urinary erythropoietin: preparation with high potency.

Human urinary erythropoietin has been purified to homogeneity. The seven-step procedure yielded a preparation with a potency of 225,000 U/mg protein. SDS-polyacrylamide gel electrophoretic analysis of the purified hormone revealed a single protein band with a molecular weight of about 35,000 that migrated with the biological activity. As to its stability, the purified hormone retained its activity in the presence of 0.001% Tween 20.

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.

Partial purification of human urinary megakaryocyte colony-stimulating factor.

Urinary extracts from patients with aplastic anemia are known to promote murine megakaryocytopoiesis. In this report, we show a simple method for the partial purification of megakaryocyte colony-stimulating factor from human urine. A four-step purification procedure, which included ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-Sepharose chromatography and high-resolution hydroxyapatite chromatography, resulted in an about 430- to 630-fold increase of specific activity. The final fractions were still contaminated with erythropoietin, but the contaminated content of erythropoietin was not enough to stimulate mouse megakaryocytopoiesis in our culture system. We also demonstrate that human urinary extracts stimulated human megakaryocyte colony formation.

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.