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Highly sensitive determination of plasma cytokines by time-resolved fluoroimmunoassay; effect of bicycle exercise on plasma level of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma).

A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu3+ as a powerful labeling material. The minimum detection limits of cytokines, i.e. IL-1 alpha, TNF alpha, and interferon gamma (IFN gamma) were about 1/10 smaller than those of enzyme-linked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1 alpha, TNF alpha, and IFN gamma were determined. In the case of incremental exercise, IL-1 alpha increased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN gamma increased in recovery period, 2 h after the light-exercise. TNF alpha level was significantly higher in a heavy-exercise. The concentration of IL-1 alpha peaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.

Adult↗

Effects of hypothyroidism and withholding of feed on plasma lipid concentrations, concentration and composition of very-low-density lipoprotein, and plasma lipase activity in horses.

OBJECTIVE: To evaluate selected concentrations of blood lipids and lipase activities in euthyroid and hypothyroid horses deprived of feed for 96 hours. ANIMALS: 4 healthy adult mares and 4 thyroidectomized adult mares. PROCEDURE: Horses were deprived of feed for 96 hours. Blood samples were collected at 24-hour intervals and analyzed to determine concentrations of non-esterified fatty acid (NEFA), triglyceride (TG), total cholesterol (TC), and very-low-density lipoprotein (VLDL) as well as composition of VLDL. Plasma lipase activities were measured after feed was withheld for 96 hours and 12 days after resumption of feeding. RESULTS: Time significantly affected plasma NEFA, VLDL, TG, and TC concentrations in both groups of horses. During the 96-hour period, mean plasma concentrations of NEFA and VLDL increased 10-fold in euthyroid horses and increased 5-fold and 9-fold, respectively, in hypothyroid horses. Mean plasma TG concentrations increased 8-fold in both groups, and plasma TC concentrations significantly increased by 33 and 30%, respectively. Composition of VLDL was significantly affected by feed deprivation in euthyroid horses. Activities of lipoprotein lipase and hepatic lipase were significantly higher in feed-deprived horses. Activity of hepatic lipase was significantly lower in hypothyroid horses than in euthyroid horses. CONCLUSIONS AND CLINICAL RELEVANCE: Hypothyroidism did not significantly alter the magnitude of the response of blood lipids to feed deprivation. Thyroid hormones may reduce variability in blood lipid concentrations but do not determine susceptibility to hyperlipemia. Hypothyroidism does not appear to be a factor in the pathogenesis of hyperlipemia in horses.

Animals↗

The effects of high-forage diets with added palm oil on performance, plasma lipids, and carcass characteristics of ram lambs with initially high or low plasma cholesterol.

The objectives of this study were to examine the interaction between added palm oil in high-forage diets and initial concentration of plasma cholesterol on performance, plasma lipids, and carcass characteristics of growing ram lambs. Thirty-two Hampshire-Suffolk ram lambs (initial BW = 34.4 kg) were assigned to a 2 x 2 factorial design consisting of diet (basal [NPO] or 10.7% added palm oil [PO]) and initial plasma cholesterol concentration (high mean = 50 mg/dL [HC] or low mean = 38 mg/dL [LC]; SEM = 2; P = .01). The lambs were individually fed diets (77% forage-23% concentrate) that contained 16.0% CP, 2.14 Mcal of ME/kg (NPO), and 2.62 Mcal of ME/kg (PO). Metabolizable energy intakes were adjusted to .20 Mcal/kg of BW.75 for both dietary treatments. Lambs were weighed and feed intakes adjusted weekly. Lambs were bled via jugular venipuncture on d 28, 56, and 84 and lambs were slaughtered after they had been fed the diets for 90 d. Plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, triglycerides, and nonesterified fatty acids were increased (P = .01) by feeding PO. Lambs fed PO were fatter than lambs fed NPO, as indicated by greater subcutaneous fat thickness and kidney and pelvic fat. Initial plasma cholesterol concentration had little effect on any of the parameters measured. Lambs fed PO had fatter carcasses than lambs fed NPO at calculated equalized ME intakes, which indicates that energy deposition is more efficient in palm oil-supplemented diets.

Animal Feed↗

Equine dysautonomia (grass sickness) is associated with altered plasma amino acid levels and depletion of plasma sulphur amino acids.

To determine whether equine dysautonomia (ED) is associated with alterations in plasma amino acid metabolism, plasma amino acid profiles were determined for horses with acute (n = 10), subacute (n = 6) and chronic (n = 7) ED and for healthy cograzing horses (n = 6) and control horses (n = 10). Horses with acute ED had perturbations in plasma amino acid profiles resembling those of severe protein malnutrition. In addition, horses with ED and cograzing healthy horses had depletion of the plasma sulphur amino acids cyst(e)ine and methionine. As similar plasma amino acid perturbations occur in subacute/chronic cyanide toxicity, the role of cyanogenic glycosides in the aetiology of ED warrants further study. Unfortunately, amino acid analysis cannot be used as a definitive premortem diagnostic test for ED, since there was overlap in the individual amino acid levels of control, cograzing and ED horses.

Acute Disease↗

Plasma mineralocorticoids, plasma renin, and urinary kallikrein in salt-sensitive and salt-resistant rats.

Plasma aldosterone, deoxycorticosterone (DOC), 18-hydroxy-deoxycorticosterone (18OH-DOC), and corticosterone were measured in Dahl salt-sensitive (S) and salt-resistant (R) rats. Plasma corticosterone and DOC were not different between strains but plasma aldosterone was decreased and plasma 18OH-DOC increased in S compared to R. Plasma renin activity and urinary kallikrein excretion were both lower in S than R. Urinary kallikrein is known to vary directly with mineralocorticoid activity and 18OH-DOC is a weak mineralocorticoid. The lower urinary kallikrein in the presence of elevated 18OH-DOC could mean that urinary kallikrein differences between S and R are under independent genetic control from 18OH-DOC.

18-Hydroxydesoxycorticosterone↗

Plasma osteocalcin in preparturient and postparturient cows: correlation with plasma 1,25-dihydroxyvitamin D, calcium, and inorganic phosphorus.

The purpose of the present study was to determine changes of plasma osteocalcin levels in periparturient cows and to examine the correlation of plasma osteocalcin level with that of 1,25-dihydroxyvitamin D, Ca, and inorganic P. Five Holstein-Friesian cows, aged 3 to 5 yr, were used from 5 d before to 15 d after calving. Concentration (mean +/- SE) of plasma osteocalcin decreased rapidly from d 1 prepartum, reached a low of 8.0 +/- 3.0 ng/ml at d 1 postpartum, and then recovered gradually to 17.8 +/- 3.8 ng/ml at 15 d after calving. In contrast, the concentration of plasma 1,25-dihydroxyvitamin D increased from calving to 3 d postpartum. Plasma concentrations of osteocalcin did not correlate significantly with those of 1,25-dihydroxyvitamin D, but it was significantly proportional to that of Ca and inorganic P. It is suggested that osteoblast function is depressed by a number of factors around the time of parturition.

Animals↗

Decreased plasma proteins, increased total plasma-free amino acids, and disturbed amino acid metabolism in the hereditary severe anemia of the Belgrade laboratory (b/b) rat.

The plasma amino acid pattern has been investigated in severely anemic Belgrade laboratory (b/b) rats. Nonanemic heterozygous (b/+) or normal homozygous (+/+) rats of the same age (six weeks) were used as controls. Decreased plasma proteins, increased total free amino acid, and urea concentrations in plasma associated with increased urea and 3-methylhistidine urinary excretion were found, indicating protein and amino acid metabolic alterations in anemic b/b rats. Plasma alanine, glutamine, tyrosine, and phenylalanine concentrations were increased. The significantly reduced molar ratio between valine+leucine+isoleucine and phenylalanine+tyrosine suggested severe disturbance in the hepatic energy-producing system and derangement of hepatic energy status. Partial or complete reversal of the anemia within 3 days by red blood cell transfusion or within 3 weeks by iron treatment resulted in normalization of tyrosine, alanine, glutamine, and total amino acid concentrations in plasma, as well as of molar ratio between valine+leucine+isoleucine and phenylalanine+tyrosine. This indicated a better oxygen supply to the liver and normalization of the hepatic energy status. These findings suggest that the metabolic disturbances in the b/b rat are the consequence of hypoxia due to the severe anemia.

Alanine↗

Nutritional regulation of plasma tumor necrosis factor-alpha and plasma and urinary nitrite/nitrate responses to endotoxin in cattle.

Effects of dietary protein level with and without L-arginine (Arg) infusion on plasma tumor necrosis factor-alpha (TNF-alpha) response to endotoxin (lipopolysaccharide [LPS]) as well as plasma concentration and urine output of nitrite and nitrate (NOx), the stable end products of nitric oxide radical (NO), were studied in beef heifers (275-310 kg body wt). The animals were fed low- (LP; 7.96%) or high- (HP; 13.94%) protein diets for 10 days before LPS administration (Escherichia coli; 0.2 microgram/kg, iv). L-Arginine in saline (0.5 g/kg body wt) or saline was infused for 8 hr with one-third of total Arg infused before LPS administration. Plasma TNF-alpha concentrations increased in all heifers after LPS injection (peak at 1 hr and return to baseline at 4 hr); however, concentrations were lower in HP- than in LP-fed heifers at 1, 2, and 3 hr. Infusion of Arg did not affect plasma TNF-alpha response to LPS. Plasma NOx concentrations increased in all heifers after LPS challenge; compared with saline, Arg infusion increased the total response (integrated area under concentration curve) in LP- but not in HP-fed heifers. Relative to pretreatment period, the rate of NOx output in urine collected 2-6 hr after LPS administration increased in all heifers regardless of dietary protein level and was further amplified by Arg infusion. The rate of NOx output in urine collected 6-24 hr after LPS challenge was even higher in LP-fed heifers infused with Arg but returned to the basal values in other groups. Activity of hepatic inducible NO synthase was not affected by LPS, Arg, or dietary protein level at the time points studied. The data suggest that dietary protein levels can modulate both TNF-alpha and NO responses to LPS in cattle; high dietary protein intake decreases TNF-alpha response and attenuates the conversion of supplemental Arg to NO.

Animals↗

Plasma levels of estradiol and plasma protein binding of sex steroids in dogs. An investigation with special reference to development of hip dysplasia in growing individuals.

Peripheral plasma levels of estradiol were determined in 39 dogs of three breeds (Greyhound, German Shepherd, Golden Retriever). Blood samples were collected weekly from 3 weeks up to 17--21 weeks of age. The pups from all three breeds had mean levels of estradiol varying between 4--8 pg/ml. The lowest levels were found in pups which developed hip dysplasia. There was a significant difference (0.01 less than p less than 0.005) between the estradiol levels of German Shepherd pups with normal and dysplastic hip joints. In female Greyhounds, the peripheral plasma levels of estradiol were examined from 17 weeks of age through the first heat. No increase of the estradiol level was seen with increasing age. The ability of dog plasma to bind dihydrotestosterone (DHT) and estradiol was examined in three dogs. It was found that there are at least three types of binding components for DHT: one with high affinity and extremely low capacity, which is heat labile, another which is also heat labile but has a high capacity and low affinity, and a third which appears to be albumin. Estrogen administration to the three dogs lowered the binding of DHT. It was concluded that it is most unlikely that hyperestrogenism is an etiologic factor in canine hip dysplasia. Unphysiologically high doses of estradiol have been used for experimental induction of hip dysplasia. There is a possibility that the levels of plasma proteins, which bind steroids, could be of importance for the etiology of hip dysplasia. A low plasma level of estradiol could be biologically highly significant if there is a low level of a specific binding protein.

Animals↗

Determination of a new asiatic acid derivative, AS 2-006A in rat plasma and urine, and human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a new wound healing agent, AS 2-006A (ethoxymethyl 2-oxo-3, 23-O-isopropylideneasiatate), in rat plasma and urine, and human plasma. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto the reversed-phase column. The mobile phase employed was acetonitrile : H2O (9:1, v/v) and run at a flow rate of 1.1 ml/min. The column effluent was monitored by a UV detector set at 205 nm. The retention time for AS 2-006A was approximately 29.5 min. The detection limits for AS 2-006A in rat and human plasma were both 1 microg/ml, and in rat urine was 2 microg/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 10.8%) for rat plasma and urine, and human plasma. No interferences from endogenous substances were found.

Animals↗

[Chronic peripheral arterial occlusive disease, platelet glycoproteins GPIIb-IIIa and GP Ib-IX, plasma von Willebrand factor and plasma fibrinogen concentrations in patients with type 2 diabetes mellitus].

Diabetes mellitus (DM) type 2 is a very strong risk factor for atherosclerosis. The final event of atherosclerosis is the vessels occlusion by platelet riche thrombus. Platelets adhesion and aggregation is mediated by interaction between platelets glycoproteins: GPIb-IX, GPIIb-IIIa and adhesive proteins: von Willebrand factor or fibrinogen. The expression of platelets GPIb-IX, GPIIb-IIIa, plasma vWF, fibrinogen concentrations were evaluated in 40 patients with diabetes type 2 (22 patients with PAOD stage II and IV according to Fontain, 18 diabetics without paod) and 32 healthy individuals. The expression of platelets glycoproteins GPIIb-IIIa and GPIb-IX was estimated by ELISA using monoclonal antibody against GPIIb-IIIa (CD41a) and GPIb-IX (CD 42a Immunotech). Plasma vWf (189.7 +/- 53.6%), fibrinogen (4.5 +/- +/- 1.3 g/l) level and expression of platelets GPIb-IX (63.2 +/- 19.6% in platelets concentration 125,000/mm3, 104.5 +/- 28.1% in platelets concentrations 250,000/mm3) and GPIIb-IIIa 50.8 +/- 10.1% in platelets concentrations 125,000/mm3, 95.3 +/- 21.3% in platelets concentrations 250,000/mm3 were statistically higher in patients with diabetes type 2 than in controls (vWf: 94.9 +/- 27.1%, fibrinogen: 2.8 +/- 0.4 g/l, GPIb-IX in platelets concentration 125,000/mm3: 43.8 +/- 9.3%, in concentration 250,000/mm3: 83.9 +/- 18.3%, GPIIb-IIIa in platelets concentration 125,000/mm3: 33.7 +/- 10.1%, in platelets concentration 250,000/mm3: 63.2 +/- 15.4%). We found significant correlation between the expression of GPIIb-IIIa, GPIb-IIIa, GPIb-IX and plasma adhesive proteins: vWF, fibrinogen in controls and both subgroups of diabetic patients. The correlation between plasma vWF and fibrinogen level and degree of arterial insufficiency in diabetic patients was also found. We can assume that higher vWf, fibrinogen plasma level in diabetic patients with and without PAOD could account for high expression of platelets GPIIb-IIIa and GPIb-IX.

Adult↗

Plasma concentrations of diazepam and its metabolites after peroral, intramuscular, and rectal administration. Correlation between plasma concentration and sedatory effect of diazepam.

Plasma levels of diazepam, N-demethyldiazepam and free oxazepam were measured gaschromatographically in ten healthy volunteers after 5 mg of diazepam perorally, intramuscularly and rectally (with three different kinds of suppositories). The best absorption of diazepam was found after peroral administration. After an intramuscular injection a delayed absorption with low plasma concentrations of diazepam was found. The basal component of a diazepam suppository seems to have a great effect on the rectal absorption of diazepam. Two of the three different kinds of diazepam suppositories caused higher plasma diazepam concentrations than the intramuscular injection of the drug. There were no great differences in the amount of the metabolites of diazepam after different kinds of administration. The subjective sedatory effect of diazepam lasted approximately as long as the fast distribution of diazepam from plasma took place. A very highly significant correlation between plasma concentration and subjective sedatory effect of diazepam after a single dose was found.

Administration, Oral↗

[Plasma- and tissue concentrations following intramuscular administration of etofenamat. Pharmacokinetics of etofenamat and flufenamic acid in plasma, synovium, and tissues of patients with chronic polyarthritis after administration of an oily solution of etofenamat].

Studies on Plasma and Tissue Concentrations of Etofenamate following Intramuscular Application/Pharmacokinetics of etofenamate and flutenamic acid in plasma, synovia and tissues of patients with chronic polyarthritis after application of oily etofenamat solution Pharmacokinetics of etofenamate (ETO, CAS 30544-47-9; Rheumon i.m.) and flufenamic acid (FLU, CAS 530-78-9) were investigated in plasma, synovial fluid, and tissues after single intramuscular application of etofenamate to patients with rheumatoid arthritis. 62 patients with indicated operative procedure in the knee-joint received a single dose of etofenamate dissolved in oil before operation. At definite times between 1.5 and 48 h post injectionem samples from 6 patients of each time group were collected. Samples of plasma, synovial fluid, synovial membrane, muscle, bone, hyaline cartilage, and fat tissue and in some cases meniscus cartilage were taken. Concentrations of ETO and its active metabolite, FLU, were determined by HPTLC. In all tissues investigated, concentration/time courses of ETO and FLU were observed. ETO and FLU were measured first in all matrices 1.5 h at the latest 3 h post injectionem. Pharmacokinetics in tissues follows that in plasma. Rate-limiting step is the liberation of drug from the oil depot. For a long period pharmacokinetics of ETO and FLU is mainly determined by the constant liberation from the oil depot (zero order kinetics of liberation). Zero order kinetics is deduced from the linear ascent of the cumulated AUC (in percent) vs. time plot. It is directly related to the liberation of drug from the galenical formulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

In vitro plasma perfusion through adsorbents and plasma ultrafiltration to remove endotoxin and cytokines.

In vitro plasma perfusion experiments were performed using small columns containing either resin or charcoal adsorbents to assess the removal of cytokines and endotoxin. 125I-labelled tumor necrosis factor-alpha (TNF-alpha; 500 pg/ml) and interleukin-6 (IL-6; 10 ng/ml) were added individually to human plasma. Over 4 hr of perfusion, Amberlite XAD-7 resin removed 32.5% +/- 3.3% (n = 5) of the initial amount of TNF-alpha and 71.4% +/- 3.8% (n = 5) of the initial amount of IL-6. DHP-1 polyhema-coated activated charcoal removed 17.2% +/- 6.2% (n = 5) of TNF-alpha and 48.5% +/- 7.4% (n = 5) of IL-6. Preliminary experiments were performed with lipopolysaccharide (LPS; 100 ng/ml) and interleukin-1 alpha (IL-1 alpha; 500 pg/ml), which showed that, over 4 hr, Amberlite XAD-7 removed 10.3% of the initial LPS and 29.1% of IL-1 alpha, whereas DHP-1 charcoal removed 23.2% of the initial LPS and 65.3% of IL-1 alpha. In vitro plasma ultrafiltration with either polysulfone or polyacrylonitrile membranes, as used clinically in haemodialysis, was performed with recirculation of plasma containing LPS or TNF-alpha. Neither of the substances was filtered to a significant degree. In conclusion, direct removal of these inflammatory mediators from the circulation of patients with multiorgan failure due to fulminant hepatic failure or sepsis would be possible by perfusion of plasma through adsorbents but not by haemodialysis.

Adsorption↗

Plasma precursors of estrogen. III. Conversion of plasma dehydroisoandrosterone to estrogen in young nonpregnant women.

The utilization of plasma dehydroisoandrosterone for estrogen production was studied in 4 normal young nonpregnant women and in one young surgical castrate woman. The mean transfer constant for total conversion to estrogen, [sigma] DE-BU' was 0.0016 of which about one third (0.0005) could be accounted for by conversion of dehydroisoandrosterone first to plasma androstenedione which in turn was converted to estrone. The remaining fractional conversion appeared to result principally in estradiol formation via a pathway probably not involving plasma androstenedione. The conversion of plasma dehydroisoandrosterone to estrogen was similar in the surgical castrate to that observed in the ovulating women. It is concluded that plasma dehydroisoandrosterone is converted to estrogen at extraglandular site(s) but this contribution represents only a minor fraction of total estrogen production in normal young women.

Androstenedione↗

Effects of age and posture on plasma active renin and plasma inactive renin in normal subjects.

To investigate the effects of age and posture on plasma active and inactive renin, we measured the plasma active renin concentration (ARC) and inactive renin concentration (IRC) in 81 healthy subjects. The subjects were divided into five groups according to age and body position at the time the blood was taken. Group I included 15 five-day-old newborns in a supine position. Group H included 18 adults, aged from 20 to 50 years, who were in a supine position. Group III included 21 adults, over 50 years old, who were in a supine position. Group IV included 20 adults, aged from 20 to 50 years, who were in an upright position. Group V included 19 adults, over 50 years old, who were in an upright position. Twelve subjects were included in Groups II and IV. Plasma active renin was measured by the amount of angiotensin I general when an exogenous renin substrate was added. Plasma inactive renin was activated by trypsin. The results showed that, in a supine position, both ARC and IRC were significantly higher in newborns (Group I) than in the two adult groups (Groups II and III). The mean of the ARC/TPRC (total plasma renin concentration) ratio was lower in adults over 50 years old (Group III) than in those from 20 to 50 years old (Group II), but the difference was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Assessment of liver copper status in cattle from plasma copper and plasma copper enzymes.

Copper status determined by liver biopsy was also assessed by 3 plasma parameters: total plasma copper (Cu) as well as ceruloplasmin (Cp) and amine oxidase E.C.1.4.3.6. (AO) activities. While 14 out of the 98 cattle, all kept in field conditions, had liver copper levels of less than 20 mg/kg only 5 of these could be distinguished by low plasma Cu and 3 by low Cp activity. In addition, some animals with apparently adequate liver copper had relatively low plasma Cu values, ca 0.5-0.6 mg/l. None of the cattle had decreased AO activity. When all 3 plasma parameters were combined in the form [AO] divided by ([Cp] x [Cu]), detection of the low liver copper was improved in that 9 out of 14 could be distinguished, whereas animals with more than 20 mg/kg DM liver Cu could not. It is suggested that this relationship, tentatively called an index, would merit examination in other situations, since it might provide a better guide to copper status and indicate whether copper repletion therapy was worthwhile.

Amine Oxidase (Copper-Containing)↗

[Concentrational changes of mannose-binding proteins in blood plasma during noncomplicated pregnancy and in blood plasma of pregnant women with some infections].

Value of mannose-binding proteins was determined in plasma of pregnant women with no complications and also in pregnant women with some infections using affinitive chromatography. It was found that the concentration of mannose-binding proteins in blood plasma of women equals 0.152+/-0.025 mg/ml. The concentration of mannose-binding proteins in blood plasma increase during the consecutive trimesters of noncomplicated pregnancy. In the first trimester it equals 0.198+/-0.032 mg/ml, in the second 0.257+/-0.027 mg/ml and in the third trimester 0.345+/-0.034 mg/ml. We also found that the concentration of mannose-binding proteins in blood plasma of pregnant woman suffering with chlamydial infection equals 1.025+/-0.115 mg/ml, and in blood plasma of pregnant woman suffering with cytomegalovirus infection 1.278+/-0.144 mg/ml. The obtained data confirm a hypothesis of increased activity of innate immune system during pregnancy. Also according to this results complications accompanied by chlamydial and cytomegalovirus infections during pregnancy is the result of increasing activity of mannose-binding proteins.

Adult↗