Dipeptidyl peptidase III and alanyl aminopeptidase in the human seminal plasma: origin and biochemical properties

Human seminal plasma contained two distinct enzyme activities hydrolysing ArgArgNA. The enzymes were separated by anion exchange chromatography and further purified by gel filtration and/or hydrophobic interaction chromatography. The enzyme eluting at the lower NaCl concentration (0.26 mol/l) displayed an optimum at pH 5.7-6.0 (enzyme A), while the other enzyme eluted at 0.32 mol/l NaCl and showed an optimum at pH 8.5-9.0 (enzyme B). Enzyme A was found to coelute with an aminopeptidase which hydrolysed various amino acid derivatives as well as dipeptide naphthylamides sequentially. Both enzymes were sensitive to heavy metal ions (Cd, Cu, Hg, Pb) and chelating agents (EDTA, o-phenanthroline) and moderately sensitive to di-isopropylfluorophosphonate (DFP) or phenylmethylsulfonylfluoride (PMSF). After EDTA suppression both activities were partially reactivated by divalent metal ions, particularly by Co2+. Enzyme A was highly sensitive to amastatin, bestatin and puromycin, while enzyme B was not markedly influenced. With different substrates the modifier characteristics of enzyme A were equal. High concentrations of some substrates suppressed the hydrolysis rates of both enzymes. Enzyme B was much more sensitive to the thermal treatment than enzyme A. Tentative molecular masses of 110 kD and 80 kD were obtained for enzymes A and B, respectively. Enzyme B was found in all male reproductive tissues (testis, epididymis, vas deferens, ampulla, seminal vesicles, prostate), while enzyme A was only detected in the prostatic homogenate. Thus, ArgArgNA in the human seminal plasma is hydrolysed by dipeptidyl peptidase III, which may originate from different reproductive organs, while the prostate is responsible for the secretion of an aminopeptidase with a wide substrate spectrum including dipeptidyl derivatives.     

Diagnostic significance of type IV collagen and hyaluronic acid in the serum of patients with chronic hepatitis C for staging hepatic fibrosis

AIM: To evaluate diagnostic value of serologic fibrosis markers (hyaluronic acid--HA and type IV collagen C-IV) in patients with chronic hepatitis C (CHC) and hepatic cirrhosis (HC). MATERIAL AND METHODS: HA and C-IV were measured in 88 CHC patients with fibrosis stage 1 (n = 63) and 3 (n = 25), 13 patients with acute hepatitis C (AHC), 28 patients with hepatic cirrhosis (HC), 19 patients with pulmonary fibrosis (PF). The control group consisted of 32 healthy subjects. RESULTS: HA concentrations in the serum of CHC patients with mild to severe inflammation and fibrosis (F1 and F3) were normal (100 ng/ml). For HC diagnosis, HA test proved highly sensitive and specific (in HA 100 ng/ml sensitivity was 100%, specificity 84.6%), but this method cannot stage hepatic fibrosis. HA test was inferior to C-IV test. A mean C-IV concentration in the serum of CHC patients at the stage of marked fibrosis (F3) is significantly higher than in F1, in HC (A) patients higher than in patients with CHC F3. CONCLUSION: It is shown than concentration of C-IV above 196 ng/ml can differentiate fibrosis stage 1 from stage 3 with specificity 58.7 and sensitivity 88%.     

乙型肝炎病毒感染患者血清MBL浓度测定及临床意义

目的 探讨乙型肝炎病毒(HBV)感染患者血清中甘露糖结合凝集素(MBL)浓度变化及意义。方法 检测250例HBV感染者(病例组)及150例对照组的MBL浓度,并比较分析二者的差异。结果 病例组MBL浓度高于对照组(t=7.097,P<0.01)。病例组中高载量组MBL浓度与对照组比较差异有统计学意义(t=7.179,P<0.01),低载量组MBL浓度与对照组比较差异有统计学意义(t=4.404,P<0.01)。结论 检测HBV感染患者血清MBL浓度的变化对了解病情,观察疗效有重要的临床价值。     

High-performance liquid affinity chromatography: rapid immunoanalysis of transferrin in serum

We describe a new method for quantitatively measuring substances of clinical interest by high-performance liquid affinity chromatography (HPLAC). As a model system we selected analysis for transferrin in human serum with immobilized antibodies in a high-performance liquid chromatographic system. SelectiSpher-10 Activated Tresyl columns (5 or 10 x 0.5 cm) were used for in situ coupling of polyclonal antibodies to transferrin. The amount of transferrin eluted was determined by integrating the eluted peak at 280 nm. The whole analytical procedure--including injection of sample, washing, elution, and analysis of data--takes only 7 min. We characterized the HPLAC system for analysis of transferrin in several ways: intra-assay CV approximately 3%; inter-assay CV 2-9%; linear response up to 1 mg/mL column volume; detection limit approximately 3 micrograms; analytical recovery 98% +/- 2%; purity of eluted sample greater than 95% (SDS-PAGE). The HPLAC method was compared with "rocket" immunoelectrophoresis, a commonly used method of analysis for transferrin, and there was excellent correlation between the two methods (r = 0.96, n = 60). Benefits of this HPLAC technique include high precision, rapid analysis, and simplified sample handling.     

Effect of hepatic failure toxins on regenerative enzymes in the liver after injury with galactosamine in the rat

Hepatic thymidine kinase (TK) and ornithine decarboxylase (ODC) activities were used to quantify the regenerative response to injury with galactosamine. After massive damage with 1000 mg/kg galactosamine, TK activity (DNA synthesis) peaked between 62 and 120 hours. This peak of activity was depressed as much as 91% by six subcoma doses of dimethyl disulfide (DMDS) given between 24 hours and 64 hours after galactosamine administration. Similar doses of octanoic acid (OA) had no effect, and doses of NH4Cl had no effect except at 120 hours. The first peak of ODC activity (initiation of cell growth) at 45 hours was depressed about 60% by six subcoma doses of NH4Cl or DMDS injected between 27 hours and 42 hours. OA again had no effect. After 400 mg/kg galactosamine, a narrow but high peak of TK activity occurred at 62 hours. This peak of activity was depressed more than 50% by six subcoma doses of NH4Cl, OA, or DMDS given between 24 hours and 54 hours. The first peak of ODC activity at 36 hours was similarly reduced by more than 50% by similar doses of each of the toxins given between 24 hours and 34 hours. The overt neurologic effects of the toxins were dissipated within 1 hour of each injection. The depressive effect of NH4Cl and OA on TK and ODC activities during regeneration after massive centrolobular injury with acetaminophen was more consistently present and more extensive than that seen after injury with galactosamine.     

Direct analysis of the lectin reactivity of alpha-fetoprotein in maternal serum by crossed affinity radio-immunoelectrophoresis

Affinity experiments with the lentil (Lens culinaris) lectin have revealed the existence of two distinct molecular populations of alpha-fetoprotein: lectin reactive and lectin non-reactive. Using a combination of crossed lectin immunoelectrophoresis and radio-immunoelectrophoresis, it has been possible to obtain directly the lentil lectin affinity patterns of alpha-fetoprotein present in maternal sera. The lentil lectin reactivity of maternal alpha-fetoprotein decreases almost linearly with the gestational age from week 15 to 35.     

血浆置换对重型病毒性肝炎患者血清细胞因子的影响

目的 :观察重型病毒性肝炎患者血清细胞因子的变化 ,探讨血浆置换疗法对患者的影响。方法 :随机选择 4 0例经血浆置换治疗的重型病毒性肝炎患者 ,测定治疗前后血清肿瘤坏死因子 α(TNFα)和白介素 4(IL 4 )的水平 ,观察其与预后、肝功能、并发症间的关系。结果 :4 0例患者经治疗后 TNFα由 (79.32±2 2 .39) ng/L 下降到 (2 0 .0 1± 2 2 .2 5 ) ng/L(P<0 .0 0 1) ,IL 4由 (0 .6 1± 0 .0 7) ng/L 降至 (0 .5 7± 0 .0 6 ) ng/L(P<0 .0 1) ,好转出院的患者较恶化出院的患者下降尤其明显 (P均 <0 .0 5 ) ;肝功能的恢复同样是好转出院的患者较恶化出院的恢复得更好 (P <0 .0 5或 P <0 .0 1) ;对嗜睡、定向能力均有明显改善 (P<0 .0 1和 P <0 .0 5 ) ,对治疗各种并发症更好 (P<0 .0 1)。结论 :血浆置换能清除 TNFα和 IL 4 ,是防治各种并发症的有效措施。预后不佳者与炎症的持续存在导致血清中 TNFα和 IL 4水平较高有关。     

Effect of ursodeoxycholic acid on biochemical parameters,hepatocyte proliferation and liver histology in galactosamine hepatitis in the rat

The effect of oral administration of ursodeoxycholic acid (UDCA) on biochemical parameters, liver histology and liver cell proliferation was investigated in rats with galactosamine hepatitis. Treatment with UDCA led to a decrease of aminotransferases, but did not show any significant changes in liver histology or liver cell proliferation. The improvement of liver enzymes without change of histology in this animal model of hepatitis following treatment with UDCA is in agreement with results obtained from clinical trials with UDCA in patients with chronic viral hepatitis.     

Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients

BACKGROUND: Glycoproteins are often associated with cancer and are important in serum studies, for which glycosylation is a common posttranslational modification. METHODS: We used multilectin affinity chromatography (M-LAC) to isolate glycoproteins from the sera of breast cancer patients and controls. The proteins were identified by HPLC-tandem mass spectrometry (MS/MS) analysis of the corresponding tryptic digests. We used the FuncAssociate Gene Ontology program for association analysis of the identified proteins. Biomarker candidates in these groups were comparatively quantitated by use of peak area measurements, with inclusion of an internal standard. We analyzed data for concordance within the ontology association groups for vector of change with the development of breast cancer. RESULTS: Detection of the known low-concentration biomarker HER-2 (8-24 mug/L) enabled us to establish a dynamic range of 10(6), relative to the amount of albumin, for the depletion step. We then used ELISA to confirm this range. Proteins associated with lipid transport and metabolism, cell growth and maintenance, ion homeostasis, and protease inhibition were found to be differentially regulated in serum from women with breast cancer compared with serum from women without breast cancer. CONCLUSIONS: M-LAC for isolation of the serum glycoproteome, coupled with liquid chromatography-MS/MS and the use of gene ontology associations, can be used to characterize large panels of candidate markers, which can then be evaluated in a particular patient population.     

Thyroid function in hospitalized patients: effect of illness and serum albumin concentrations

We assessed the clinical utility of measuring thyrotropin (TSH) in serum by immunoradiometry and of measuring total thyroxin (TT4), total triiodothyronine (TT3), free thyroxin (FT4), and free triiodothyronine (FT3). We used a group of 110 healthy volunteers, 45 ill hypoalbuminemic patients, and 42 ill normoalbuminemic patients. In addition, the free thyroxin index (FTI) and TT4:thyroxin-binding globulin (TBG) ratio were also calculated. The hypoalbuminemic group had significantly lower FT4, FT3, TT4, TT3, and FTI concentrations, but only FT3 and TT3 were significantly lower in the ill normoalbuminemic group as compared with controls. We found significant correlation between FT4 and albumin (r = 0.372, P less than 0.001) and FT3 and albumin (r = 0.465, P less than 0.001). TSH concentrations were undetectable in two of 45 hypoalbuminemic patients, significantly higher in the rest. The TT4/TBG ratio was the only parameter of thyroid function that remained unchanged in the ill patients.     

An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.     

Origin of plasma lysophosphatidylcholine: evidence for direct hepatic secretion in the rat

In the plasma, lysophosphatidylcholine (LPC) is formed by the action of lecithin-cholesterol acyltransferase (LCAT) when a fatty acid is removed from plasma phosphatidylcholine (PC) and transferred to cholesterol. To determine whether plasma LPC might also be generated by the hydrolysis of hepatic PC, we assessed phospholipid production by the isolated perfused rat liver. Bile duct-cannulated livers were perfused with bile salt and a recirculating, lipid-free medium containing albumin. We found that LPC accumulated in the perfusate to a greater extent than any other phospholipid, exceeding the accumulation of PC (the second most prevalent phospholipid) twofold. We further found that perfusate LPC was not formed by hydrolysis of PC in the perfusate and was not dependent on the presence of infused bile salt. LPC that accumulated in the perfusate was highly unsaturated and markedly dissimilar to the more saturated LPC that results from the activity of LCAT. Results thus indicate that the isolated liver directly secretes LPC, which is presumably generated from hydrolysis of hepatic PC. Because plasma LPC is to a great extent unsaturated in the live rat, these findings suggest that direct hepatic secretion is a quantitatively important source of plasma LPC.     

慢性乙型肝炎患者血清e抗原及病毒载量和肝损伤的相关性研究

目的观察慢性乙型肝炎(CHB)患者不同乙型肝炎e抗原(HBeAg)状态与HBV-DNA载量及肝脏损伤的关系。方法分别采用实时荧光定量PCR法及速率法对258例慢性乙型肝炎进行血清HBV-DNA定量和丙氨酸氨基转移酶(ALT)检测。以ALT水平对肝脏损伤程度进行分型,对不同HBeAg状态、病毒载量、肝损伤程度进行分析。结果 HBeAg阴性组年龄大于HBeAg阳性组,其血清HBV-DNA载量低于HBeAg阳性组(P0.01),肝损伤程度两组比较差异无统计学意义(P0.05)。HBeAg阴性组随血清HBV-DNA载量增加,肝损伤程度明显升高(P0.05),HBeAg阳性组则无相关性。结论 HBeAg阳性是判断HBV复制的良好指标,HBeAg阳性CHB患者体内DNA载量与肝内炎症损伤无关。而HBeAg阴性CHB患者体内HBVDNA与肝脏损伤相关,应重视对HBeAg阴性患者的随访和治疗。     

慢性乙肝患者血清HBV DNA含量与肝组织损伤及纤维化程度的相关性研究

目的了解慢性乙型肝炎患者血清 HBV DNA含量与肝组织损伤及纤维化程度的相关性.方法对 64例 HBsAg阳性的慢性乙型肝炎进行肝穿刺病理检查, HBV DNA定量采用荧光定量系统, HBV M采用 ELISA法.结果慢性中、重度肝炎较慢性轻度肝炎血清 HBV DNA含量有显著性差异,而慢性中、重度肝炎之间差异不显著,同样肝组织炎症分级 G1、 G2、 G3随着炎症分级的增高,其血清 HBV DNA含量也逐渐增高,而在 G4级较 G3级反而有所下降;血清 HBV DNA含量与肝组织纤维化分期无明显关系;血清 HBV DNA含量随着血清胆红素水平的升高有下降的趋势.结论血清 HBV DNA水平与肝组织损伤有一定的关系,与肝纤维化程度无明显关系.     

Affinity chromatography used in distinguishing alpha-fetoprotein in serum from patients with tumors of hepatic parenchyma and of germ cells

We used affinity chromatography on concanavalin A Sepharose to study the serum alpha-fetoprotein of 10 patients with histologically proven germ-cell tumors and 12 patients with primary liver cancer. Less than 50% of the fetoprotein from germ-cell tumors bound to concanavalin A, as compared with more than 80% of the alpha-fetoprotein from primary liver cancers.