Lower expression of the alpha2,3-sialylated fibronectin glycoform and appearance of the asialo-fibronectin glycoform are associated with high concentrations of fibronectin in human seminal plasma with abnormal semen parameters.

BACKGROUND: Sialic acid isoforms expressed on glycoconjugates are known to be involved in different biological functions. The aim of this study was to compare alpha2,3- and/or alpha2,6-linked sialic acid expression on fibronectin in the seminal plasma of male partners from infertile couples. The data obtained were analyzed in relation to normal and abnormal semen parameters, as well as to fibronectin concentrations. METHODS: Fibronectin concentrations were determined by ELISA using a specific monoclonal antibody. The relative degree of sialic acid linked to fibronectin glycans either by alpha2,3 or alpha2,6 isomeric linkage was estimated by lectin-fibronectin ELISA utilizing lectins from Maackia amurensis and Sambucus nigra, respectively. RESULTS: High seminal fibronectin concentration was more frequently associated with abnormal semen parameters. The glycans of seminal plasma fibronectin, in contrast to blood plasma fibronectin, contained sialic acid more frequently attached by alpha2,3 than by alpha2,6 isomeric linkage. The relative amounts of both alpha2,3- and alpha2,6-linked sialic acid fibronectin isoforms were lower in the seminal plasma of men suspected of infertility. CONCLUSIONS: Seminal fibronectin showed an oncofetal type of sialylation and the distribution of hypo- and asialylated fibronectin glycoforms were associated with abnormal semen parameters and with high concentrations of fibronectin.     

Fucosylation of alpha1-acid glycoprotein (orosomucoid) compared with traditional biochemical markers of inflammation in recent onset rheumatoid arthritis

BACKGROUND: Fucosylation of alpha1-acid glycoprotein (AGP, orosomucoid) has previously been found to be increased in patients with rheumatoid arthritis. Furthermore, the degree of fucosylation has been suggested to reflect disease activity. Therefore, we investigated the fucosylation of AGP in 131 patients (96 women and 35 men) with recent onset rheumatoid arthritis (RA). We compared the results with traditional biochemical markers of inflammation, i.e. plasma concentrations of AGP (P-AGP), and C-reactive protein (P-CRP). METHODS: AGP fucosylation measured with a novel lectin enzyme-linked immunosorbent assay (ELISA) was compared with a disease activity score (DAS28) and its components, and with P-AGP, and P-CRP at the time of diagnosis, and at a follow-up visit 1 year later. RESULTS: Both men and women with RA had increased AGP fucosylation compared to healthy individuals. We found a weak correlation between AGP fucosylation and DAS28 only in men. In men with initially increased AGP fucosylation, the level of fucosylation correlated with the change in DAS28 during the first year following diagnosis. CONCLUSION: We conclude that AGP fucosylation is not superior to traditional markers of disease activity in RA. However, AGP fucosylation may give some additional information to traditional biochemical markers on the disease progression in men.     

Inflammation-induced expression of sialyl Lewis X-containing glycan structures on alpha 1-acid glycoprotein (orosomucoid) in human sera

The glycosylation of the acute phase glycoprotein alpha 1-acid glycoprotein (AGP) in human sera is subject to marked changes during acute inflammation as a result of the cytokine-induced hepatic acute phase reaction. The changes described thus far comprise alterations in the type of branching of the carbohydrate structures as revealed by increased reactivity of AGP with concanavalin A. We now report on acute inflammation-induced increases in alpha 1-->3-fucosylated AGP molecules, as detected by the reactivity of AGP towards the fucose- binding Aleuria aurantia lectin (AAL) in crossed affino- immunoelectrophoresis of human sera. Laparotomy of women, for the removal of benign tumors of the uterus, was used as a model for the development of the hepatic acute phase response. Hugh increases were detected in the amounts of strongly AAL-reactive fractions of AGP, presumably containing three or more fucosylated N-acetyllactosamine units. At least part of these Lewis X-type glycans (Gal beta 1-->[Fuc alpha 1-->3]GlcNAc-R) appeared to be substituted also with an alpha 2-- >3-linked sialic acid residue. This was revealed by the laparotomy- induced abundant staining of AGP with an antisialyl Lewis X monoclonal antibody (CSLEX-1) on blots of sodium dodecyl sulfate-polyacrylamide gels containing AGP isolated from the sera of a patient at various days after operation. It is concluded that acute inflammation induces a strong increase in sialyl Lewis X-substituted AGP molecules that persists at a high level throughout the inflammatory period. We postulate that these changes represent a physiological feedback response on the interaction between leukocytes and inflamed endothelium, which is mediated via sialylated Lewis X structures and the selectin endothelial-leukocyte adhesion molecule 1.     

Triazolam protein binding and correlation with alpha-1 acid glycoprotein concentration

On two occasions separated by a minimum of 1 wk, plasma was obtained from 12 patients (aged 18 to 73 yr) on dialysis after an overnight fast. Samples were assayed for albumin and alpha 1-acid glycoprotein (AGP) concentrations. 14C-Triazolam was added to each sample to a final concentration of 5 ng/ml. Protein binding was determined by equilibrium dialysis. Unbound triazolam ranged from 6.4% to 15.4% (mean = 10.0%). AGP concentrations ranged from 71.8 to 205.1 mg% (mean = 123.4 mg%). Triazolam binding ratio (bound/unbound concentration) correlated with AGP concentration (r2 = 0.69) but not with albumin concentration, age, or sex. This correlation was verified by adding AGP in varying amounts to control plasma.     

Altered glycosylation of alpha1-acid glycoprotein in patients with inflammation and diabetes mellitus

BACKGROUND: In certain pathophysiological conditions, such as inflammation rheumatoid arthritis and diabetes mellitus (DM), alterations in asparagine-linked glycan (N-glycan) patterns of the acute-phase protein, alpha(1)-acid glycoprotein (AGP), have been reported. In this study, we investigated N-glycan structures of AGP purified from the sera of patients with acute inflammation (n=5), type 2 diabetes mellitus (n=5), and healthy individuals (n=5). METHODS: N-Glycans were released with peptide N-glycosidase F (PNGase F) from denatured AGP and purified with cellulose cartridge. N-glycans were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) in combination with exoglycosidase digestion. RESULTS: We revealed increases in bi-antennary complex glycans and in alpha1-3 fucosylated bi-, tri-, and tetra-antennary glycans and a decrease in tri-antennary glycans in inflammation patients. These results support increases in bindings to concanavalin A (ConA) and Aleuria aurantia lectins (AALs). In diabetic patients, the pathogenesis-specific change in N-glycan patterns of AGP was not significant. CONCLUSIONS: The MALDI-TOFMS method is sensitive and suitable for profiling analysis of N-glycans in clinical samples.     

Microheterogeneity of alpha 1-acid glycoprotein in healthy elderly subjects: patterns obtained by crossed affino-immunoelectrophoresis.

Total serum alpha 1-acid glycoprotein (AGP) concentration and concanavalin A-dependent microheterogeneity were studied in 31 healthy elderly subjects (18 men, 13 women, 71 to 76 yr old). Crossed affino-immunoelectrophoresis (CAIE) revealed three microheterogeneity variants of AGP: non-reactive, weakly reactive and strongly reactive with ConA. Two patterns were found in both elderly men and women, i.e. a normal pattern and one with an increase in the non-reactive form. Mean serum AGP levels in the elderly subjects with slightly higher than in a reference group of younger subjects. The Con A non-reactive form of AGP was increased in 42% of the elderly population. An increase in the non-reactive form of AGP in CAIE should be considered as general expression of chronic inflammation which is of no clinical relevance.     

Changes in alpha 1-acid glycoprotein serum concentrations and glycoforms in the developing human fetus.

alpha 1-Acid glycoprotein concentrations and reactivity to concanavalin A were measured in maternal and fetal serum and amniotic fluid obtained from 24 women undergoing diagnostic cordocentesis at 20 to 33 wk gestation and in 30 additional fetal sera (19 to 34 weeks gestation). Maternal alpha 1-acid glycoprotein serum levels were five to ten times higher than fetal and amniotic levels. Fetal alpha 1-acid glycoprotein levels were found to increase with advancing gestational age. Using crossed immunoaffino electrophoresis with concanavalin A, alpha 1-acid glycoprotein patterns were identical in maternal serum and amniotic fluid but totally different in fetal serum. The fetal concanavalin A pattern changed progressively during fetal life towards that of the newborn. These data confirm earlier assumptions of fetal synthesis of alpha 1-acid glycoprotein and provide normal reference values for alpha 1-acid glycoprotein in fetal serum. In addition, the specific fetal concanavalin A pattern indicates that the alpha 1-acid glycoprotein glycosylation process during fetal life differs from that in post-natal life.     

Xylosyltransferase activity in seminal plasma of infertile men

BACKGROUND: Proteoglycans play an important role during the mammalian conception process and are functionally involved in the preservation of the sperm motility and velocity. Human xylosyltransferase (EC 2.4.2.26, XT) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan chains in proteoglycans. XT activity in body fluids was shown to be an indicator for the actual proteoglycan biosynthesis rate. METHODS: Xylosyltransferase activity was determined in seminal plasma and serum samples of 50 healthy semen donors, 20 infertile men with oligo-, astheno- or teratozoospermia and men after vasectomy. For identification of the XT secreting glands split ejaculates were analyzed. RESULTS: XT activity in seminal plasma samples of infertile men was significantly reduced (mean 1.53 mU/l, 90% range 0.95-2.52 mU/l, p<0.05) in comparison to healthy donors (mean 3.21 mU/l, 90% range 1.82-4.65 mU/l). The analysis of the fractionated ejaculates revealed that the highest XT activity was found in portions secreted by the seminal vesicle glands. CONCLUSIONS: In view of the increasing interest for in vitro fertilization, monitoring the seminal plasma XT activity of men with unexplained infertility is proposed to be an advantageous additional biochemical parameter that could be useful for improving the methods producing pregnancy in some couples.     

Semen polymorphonuclear neutrophil leukocyte elastase as a diagnostic and prognostic marker of genital tract inflammation--a review.

Elastase is a protease released by polymorphonuclear neutrophils (PMN) during the inflammatory process. Since 1987, seminal elastase-inhibitor complex (Ela/alpha1-PI) has been proposed as a marker of male silent genital tract inflammation. Measured by immunoassay in seminal plasma, Ela/alpha1-PI at a cut-off level of > or = 230 microg/l, is useful in the detection of genital tract inflammation. The prevalence of increased seminal Ela/alpha1-PI in infertile men is significantly higher than that observed in fertile men. The Ela/alpha1-PI level is positively correlated with other seminal fluid markers of male genital tract inflammation: reduced semen volume, citric acid, fructose, and increased albumin, complement component C3, caeruloplasmin, immunoglobulins IgG and IgA, and cytokines interleukins-8 and -6. A higher seminal Ela/alpha1-PI level is significantly associated with tubal damage in female partners. After antibiotic therapy, a decrease of Ela/alpha1-PI level is observed. The presence of tubal damage in the partner may negatively affect the response to antibiotic treatment. A higher seminal Ela/alpha1-PI is associated with lower percentage of sperm with single-stranded deoxyribonucleic acid (DNA) and better fertilization rate in in vitro fertilization. Besides infertility, the determination of Ela/alpha1-PI is useful to confirm the presence of prostate and other male accessory gland bacterial inflammation. Screening for PMN Ela/alpha1-PI is easy to perform and reproducible and is a reliable quantitative test for diagnosis and prognosis of silent genital tract inflammation of couples. Moreover, sequential determinations allow the follow-up of inflammation during and after therapy.     

Glycosylation of alpha 1-acid glycoprotein in relation to duration of disease in acute and chronic infection and inflammation.

Microheterogeneity of acute phase proteins frequently differs in acute and chronic types of inflammation. However, it is unknown whether these changes depend on the duration of the inflammation in a given disease. We therefore investigated the microheterogeneity of alpha 1-acid glycoprotein (AGP) in sera from patients with acute and chronic bacterial infection in comparison to rheumatoid arthritis and ankylosing spondylitis. In acute bacterial infection Con A-reactivity of AGP was significantly elevated. By contrast, AGP in chronic bacterial infection showed the same glycosylation pattern as rheumatoid arthritis and ankylosing spondylitis being characterized by a decreased reactivity to Con A. Serial measurements in individual patients with bacterial infections showed a transition from the initially elevated to decreased reactivity to Con A as the disease became chronic.     

Superoxide dismutase activities of spermatozoa and seminal plasma are not correlated with male infertility

Abnormal reactive oxygen species (ROS) production is associated with defective sperm function. Superoxide dismutase (SOD) is related with the scavenging of seminal ROS. We aimed to determine the effect of SOD activities of spermatozoa and seminal plasma on sperm quality. Semen samples from infertile couples who consented to the analyses were divided into two groups: 1) normospermia (n = 20); and 2) oligoasthenozoospermia (n = 31). The SOD activities of the spermatozoa and seminal plasma were measured by determining the inhibition of pyrogallol autoxidation. The SOD activities of spermatozoa and seminal plasma in both groups were compared. The relationships between the SOD activities and the sperm qualities were determined. We noted that SOD activities of sperm/seminal plasma in both groups were nonsignificantly different (group 1 vs. 2 = 0.77 +/- 0.33/0.84 +/- 0.40 U/mg protein for sperm, and 0.66 +/- and 0.36/0.83 +/- 0.47 U/ml for seminal plasma). SOD activities of sperm/seminal plasma were positively but nonsignificantly correlated with the sperm motility (SOD of sperm = 0.0008 x motility + 0.67; SOD of seminal plasma = 0.0006 x motility + 0.81) and concentration (SOD of sperm = 0.0006 x concentration + 0.67; SOD of seminal plasma = 0.0021 x concentration + 0.73). We concluded that SOD activities of sperm and seminal plasma were nonsignificantly correlated with the seminal quality. It appears that the SOD survey is not a useful tool for determining sperm fertilization potential.     

不育男性精浆中过氧化氢测定在评估精液质量中的意义

目的:探讨精浆中过氧化氢(hydrogen peroxide,H2O2)测定在评估精液质量中的应用价值。方法:收集79例男性不育患者的精液标本,按照精子密度分为A1组(精子密度≥15×106/mL)和A2组(精子密度<15×106/mL);按前向运动精子总数分为B1组(前向运动精子总数≥7.2×106)、B2组(前向运动精子总数<7.2×106);按照精子活动率分为C1组(活动率≥40%)和C2组(活动率<40%)。用比色法测定得出精浆中过氧化氢浓度,使用精子自动分析仪进行精液分析。计算出每106个精子所产生的过氧化氢含量(H2O2/精子密度),然后比较不同组之间的H2O2浓度和H2O2/精子密度有无差异。结果:(1)A1、B1和C1组中的精浆中H2O2与A2、B2、C2组相比,差异无统计学意义(P>0.05)。(2)A1、B1、C1组中H2O2/精子密度分别低于A2、B2、C2组(P<0.05)。结论:检测精浆中的H2O2可反映不育人群的精液质量,与单纯测定精浆中过氧化氢浓度相比,计算H2O2/精子密度在评估男性不育患者的精液质量方面有更高价值。     

吸烟对男性精液质量、精浆活性氧及精子凋亡率的影响

目的探索吸烟影响精液质量的机制。方法 120例男性不育患者根据吸烟习惯和烟龄分为3组：A日吸烟量〈10支,且烟龄≤1年;B日吸烟量10-20支,且烟龄5-10年;C日吸烟量≥20支,且烟龄≥10年。选用35例已生育正常健康男性作为对照。对其精液常规参数精浆MDA含量及精子凋亡率进行检测。结果不育吸烟组精子活力显著低于不育非吸烟组,精子凋亡率及精浆MDA含量显著增高;ABC三组比较,C组精子活力显著低于B组和A组,而精子凋亡率及精浆MDA含量显著增高。结论吸烟可以通过增加精浆活性氧含量及精子凋亡率,严重影响精液质量。     

男性不育患者精子形态与精浆锌和精子顶体酶活性关系的研究

目的：研究男性不育患者精子形态与精浆锌和精子顶体酶活性关系。方法455例男性不育患者分别根据年龄和精子正常形态百分率分成5组和8组,采用 Diff-Quik 染色法、改良 PRA 法和改良 Kennedy 法分别进行精子形态、精浆锌和精子顶体酶活性的检测。结果各年龄组的正常形态精子百分率和精浆锌浓度差异无统计学意义（P ＞0．05）,精子顶体酶活性在36～50岁年龄段显著降低（P ＜0．05）,其他年龄段精子顶体酶活性差异无统计学意义（P ＞0．05）。在精子正常形态百分率不同的8组中,年龄差异无统计学意义,精浆锌总量差异无统计学意义,精子顶体酶活性随着精子正常形态百分率降低而降低（r ＝0．93,P ＜0．01）。结论精子正常形态百分率不受年龄和精浆锌的影响,与精子顶体酶活性呈正相关。     

Polyclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein

This is a noncompetitive enzyme-linked immunosorbent assay for measuring low concentrations (2 to 100 micrograms/L) of human alpha 1-acid glycoprotein (AGP; orosomucoid). The method is based on a simple "sandwich" technique involving polyclonal rabbit antisera against AGP. Mean within-run and total (between-run) CVs were 6.2% and 9.7%, respectively. Analytical recovery, tested in various biological fluids, averaged 101%. The technique has been successfully applied to diluted biological fluids such as bronchoalveolar lavage, cerebrospinal and amniotic fluids, and hepatocyte-culture supernates. Because of its analytical validity and the commercial availability of the reagents, this assay is suitable for large-scale determinations of AGP concentrations in those biological fluids in which its concentration is relatively low.     

Mucuna pruriens Reduces Stress and Improves the Quality of Semen in Infertile Men

The present investigation was undertaken to assess the role of Mucuna pruriens in infertile men who were under psychological stress. Study included 60 subjects who were undergoing infertility screening and were found to be suffering from psychological stress, assessed on the basis of a questionnaire and elevated serum cortisol levels. Age-matched 60 healthy men having normal semen parameters and who had previously initiated at least one pregnancy were included as controls. Infertile subjects were administered with M. pruriens seed powder (5 g day(-1)) orally. For carrying out morphological and biochemical analysis, semen samples were collected twice, first before starting treatment and second after 3 months of treatment. The results demonstrated decreased sperm count and motility in subjects who were under psychological stress. Moreover, serum cortisol and seminal plasma lipid peroxide levels were also found elevated along with decreased seminal plasma glutathione (GSH) and ascorbic acid contents and reduced superoxide dismutase (SOD) and catalase activity. Treatment with M. pruriens significantly ameliorated psychological stress and seminal plasma lipid peroxide levels along with improved sperm count and motility. Treatment also restored the levels of SOD, catalase, GSH and ascorbic acid in seminal plasma of infertile men. On the basis of results of the present study, it may be concluded that M. pruriens not only reactivates the anti-oxidant defense system of infertile men but it also helps in the management of stress and improves semen quality.     

Changes in serum acute phase proteins in breast cancer patients receiving methotrexate infusion therapy

The serum concentrations of six glycoproteins (alpha 1-acid glycoprotein, AGP; haptoglobin, Hp; alpha 1-antitrypsin, AT; ceruloplasmin, Cp; prealbumin, PALB; and alpha 2-macroglobulin, MACRO) have been estimated serially in nine advanced breast cancer patients who received a total of 24 intravenous infusions of methotrexate (MTX). The serum glycoprotein levels taken before the first drug exposure did not relate with the prognosis of these patients. In eight patients, constantly high or rising levels of AGP, Hp and AT during consecutive infusions of the drug were associated with continued metastatic disease. A transient tumour regression occurred in one patient which correlated with falling serum levels of these proteins into the normal range. The serum levels of Cp, PALB and MACRO did not correlate with the clinical status of these patients during treatment. Possible factors which may influence the serum levels of these glycoproteins in cancer patients during therapy are discussed.     

Glycan microheterogeneity of alpha 1-acid glycoprotein in sera and dialysates of patients on continuous ambulatory peritoneal dialysis

Total concentration and concanavalin (Con A) dependent microheterogeneity of alpha 1-acid glycoprotein (AGP) were studied in sera of eight chronic renal failure patients before the start of continuous ambulatory peritoneal dialysis (CAPD), and in sera and dialysate fluids for up to 6 months of CAPD. The glycan heterogeneity of AGP in the samples was expressed as a reactivity coefficient, i.e. the ratio of the Con A-reactive AGP components to the Con A non-reactive AGP component. Concentrations of AGP in serum and reactivity coefficients were markedly elevated in non-dialysed uraemic patients. AGP concentrations in serum increased further during the first week on CAPD, and then gradually decreased to pre-dialysis values, which were reached after 1 to 6 mth. The reactivity coefficients did not change significantly during the CAPD treatment. Dialysate AGP concentrations were low in comparison to those in serum, and there was a good correlation between the reactivity coefficients in the dialysate fluids and those in the corresponding sera. The effect of peritonitis was evaluated in a separate group of eight CAPD patients. Serum and dialysate AGP concentrations were significantly higher during than after peritonitis while the corresponding reactivity coefficients were only slightly elevated.     

Distribution of primaquine in human blood: drug-binding to alpha 1-glycoprotein

To clarify the distribution of the antimalarial primaquine in human blood, we measured the drug separately in the liquid, cellular, and ultrafiltrate phases. Washed red cells resuspended at a hematocrit of 0.4 were exposed to a submaximal therapeutic level of 250 ng/ml of carbon 14-labeled primaquine. The tracer was recovered quantitatively in separated plasma and red cells. Over 75% of the total labeled drug was found in red cells suspended in saline solution, but only 10% to 30% in red cells suspended in plasma. The plasma effect was not mediated by albumin. Studies with alpha 1-acid glycoprotein (AGP), tris(2-butoxyethyl)phosphate, an agent that displaces AGP-bound drugs, and cord blood known to have decreased AGP established that primaquine binds to physiologic amounts of the glycoprotein in plasma. Red cell primaquine concentration increased linearly as AGP level fell and as the free drug fraction rose. We suggest that clinical blood levels of primaquine include the red cell fraction or whole blood level because (1) erythrocytic primaquine is a sizable and highly variable component of the total drug in blood; (2) this component reflects directly the free drug in plasma, and inversely the extent of binding to AGP; (3) the amount of free primaquine may influence drug transport into specific tissues in vivo; and (4) fluctuations of AGP, an acute-phase reactant that increases greatly in patients with malaria and other infections, markedly affect the partition of primaquine in blood. Because AGP binds many basic drugs, unrecognized primaquine-drug interactions may exist.     

Diagnostic accuracy of alpha(1)-acid glycoprotein fucosylation for liver cirrhosis in patients undergoing hepatic biopsy

BACKGROUND: Increased fucosylation of serum glycoproteins has previously been reported in patients with liver disease. We analyzed alpha(1)-acid glycoprotein (AGP) fucosylation in serum samples from patients investigated for suspected liver disease to evaluate its value as a biochemical marker for liver cirrhosis. METHODS: We used a novel lectin immunoassay adapted to the AutoDELFIA system to analyze AGP fucosylation in 261 consecutive patients admitted for liver biopsy at Malm? University Hospital in Southern Sweden. The results were compared with histopathologic findings. In addition, AGP fucosylation was compared with other biochemical markers described as useful in the diagnosis of liver cirrhosis. The biochemical markers were compared by ROC curve analysis. RESULTS: AGP fucosylation was significantly (P <0.05) higher in patients with liver cirrhosis (n = 65) than in healthy controls (n = 72), patients with normal histology (n = 29), patients with steatosis only (n = 38), patients with viral or chronic hepatitis without cirrhosis (n = 71), and patients with other liver diseases without histologic signs of cirrhosis (n = 58). By calculating the AGP fucosylation index (AGP-FI = AGP fucosylation/AGP serum concentration), we obtained a high diagnostic accuracy. The areas under the ROC curves for AGP-FI were 0.83 and 0.74 for men and women, respectively, compared with 0.82 for hyaluronic acid and 0.77 for the aspartate aminotransferase/alanine aminotransferase ratio in both men and women. CONCLUSIONS: AGP fucosylation appears to be useful in identifying patients with liver cirrhosis among patients investigated for liver disease. The lectin immunoassay showed satisfactory reproducibility and is suitable for routine use in a clinical laboratory.