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The capillary electrophoresis (CE) study was focused on quantifying glycohemoglobin A1c, HbA1c, in whole blood samples of patients suffering diabetes mellitus. The results showed that dynamic polyionic coating of the capillary made the method very reproducible. The precision evaluation, method comparison and bias estimation in CE were performed during 20 days with patient blood samples and with four control samples. The influence of the storage time and temperature on the glycohemoglobin levels were also tested. High resolution in CE could be used to show evidences of the ageing of the samples stored at -70 degrees C. The results showed that the ageing peak was not originated from HbA1c, because it did not affect the HbA1c level which was in balance with the results of fresh samples measured with immunoassay. The HbA1c % values of blood samples of 105 patients measured with the CE technique varied between 3.6 and 11.8 and they were approximately 2-3% lower than measured with immunoassay. The correlation (R2) of CE results with immunoassay and HPLC results were 0.962 and 0.781, respectively.

The title mononuclear complex, [Cu(CO(3))(C(8)H(7)N(5))(H(2)O)] x 2H(2)O, was obtained by fixation of CO(2) by a mixture of copper(II) tetrafluoroborate and the ligand bis(pyrimidin-2-yl)amine in ethanol/water. The Cu(II) ion of the complex has a distorted square-pyramidal environment, with a basal plane formed by two N atoms of the ligand and two chelating O atoms of the carbonate group, while the apical position is occupied by the O atom of the coordinating water molecule. In the solid state, hydrogen-bonding interactions are dominant, the most unusual being the Watson-Crick-type coplanar ligand pairing through two N--H...N bonds. Lattice water molecules also participate in hydrogen bonding.

Background: Current methods for determination of carbohydrate-deficient transferrin (CDT) are based on separation of the CDT fraction by ion-exchange chromatography on minicolumns and quantification by immunoassay. Alternatively, the transferrin isoforms can be separated by HPLC anion-exchange chromatography and quantified by absorbance. This method has been reported to improve the validity of CDT as a marker of chronic alcohol abuse., Methods: HPLC on either MonoQ or ResourceQ anion-exchange columns was used to separate and quantify isoforms of transferrin with detection at 460 nm. The result was expressed as the percentage of the disialo form (pI 5.7) of total transferrin (DST). The commercial CDTect assay was used as a comparison method. Serum samples from nondrinkers (n = 57), moderate drinkers (n = 77), and heavy drinkers (n = 139) were analyzed., Results: In ROC analysis for differentiation between moderate and heavy drinkers, the area under the curve (AUC) for the HPLC method was 0.87 (95% confidence interval, 0.81-0.93), whereas that for CDTect was 0.72 (95% confidence interval, 0.64-0.80). At 90% specificity, the sensitivity of DST was 63% (95% confidence interval, 53-73%) compared with 33% (22-44%) for CDT. The reference interval of the HPLC method was 0.68-1.7%., Conclusions: The HPLC anion-exchange method for quantification of CDT provides substantially better separation between moderate and heavy drinkers than the CDTect method.

Objective: To determine whether elevated homocysteine levels precede the development of preeclampsia., Methods: Study subjects were selected from a population-based cohort of 1049 nulliparous women from whom serum was collected for Down syndrome screening at 16 weeks' gestation. For 34 women who developed preeclampsia, 68 control women were chosen who remained normotensive. Homocysteine was analyzed by high-performance liquid chromatography and fluorescence detection. The sample size allowed detection of a 1.25-micromol/L difference at a significance level of 0.05 and the power of 0.81., Results: At 16 weeks' gestation, concentrations (mean, 95% confidence interval) of homocysteine in women who developed preeclampsia, 6.99 (6.42, 7.55) micromol/L, were similar to those who remained normotensive, 6.91 (6.45, 7.34) micromol/L., Conclusion: Significant changes in homocysteine metabolism did not predate the appearance of clinical preeclampsia.

Rett syndrome is a neurodevelopmental disease characterized by failure of somatic and brain growth. The insulin-like growth factor system mediates most actions of growth hormone. Evidence that it plays an important role in early development of the brain is increasing. The aim of the study reported was to assess the role of the insulin-like growth factor system in the pathogenesis of Rett syndrome. We measured insulin-like growth factor-I levels in serum (8 patients, mean age 9.1 years) and cerebrospinal fluid (13 patients, mean age 7 years) using a sensitive radioimmunoassay method and compared them with those in age-matched controls (13 and 26 patients, respectively). Neither serum nor cerebrospinal fluid insulin-like growth factor-I levels differed from those in controls. We also measured insulin-like growth factor binding protein-3 levels in serum (in 9 patients and 8 controls) and in cerebrospinal fluid (in 12 patients and 11 controls) and serum growth hormone levels (in 8 patients and 11 controls); the levels in patients did not differ from those in controls. We found no significant correlation between serum and cerebrospinal fluid insulin-like growth factor-I in Rett syndrome. This may indicate an independent role of insulin-like growth factor system in the central nervous system, making serum insulin-like growth factor-I measurement unreliable as an indicator of disturbed function in the central nervous system. Our results did not support the notion that a defective insulin-like growth factor-I system explains the lack of somatic and brain growth in Rett syndrome.

Objective: To assess the possible role of serum levels of activin A, inhibin A and pro-alpha inhibin (pro-alphaC) in insulin sensitivity in pre-eclampsia., Design: A prospective study., Setting: Helsinki University Central Hospital., Participants: Twenty-two nulliparous women with proteinuric pre-eclampsia and 16 healthy nulliparous controls in the third trimester of pregnancy., Methods: Serum samples were collected before and after intravenous injection of glucose (0.3 g/kg) and insulin (0.03 IU/kg) (the minimal model for testing insulin sensitivity), and were assayed for activin A, inhibin A and pro-alphaC., Main Outcome Measures: Comparison of the levels of activin A, inhibin A and pro-alphaC between pre-eclamptic and healthy pregnant women, and the association of these proteins with insulin sensitivity., Results: In pre-eclampsia elevated levels of activin A (139%, P = 0.0001), inhibin A (39%, P = 0.003), and pro-alphaC (92%, P = 0.0008) were observed. The amount of proteinuria (0.3-10.5 g/day) correlated positively with serum concentrations of activin A (P = 0.01) and inhibin A (P = 0.02). These glycoproteins were not associated with insulin sensitivity either in women with pre-eclampsia or controls. A 2.9-fold rise in blood glucose and a 52.5-fold rise in insulin during testing using the minimal model were not accompanied by any significant changes in activin A, inhibin A, and pro-alphaC., Conclusion: Activin A, inhibin A, and pro-alphaC are elevated in pre-eclampsia but do not appear to relate to the insulin sensitivity in pre-eclamptic or normal pregnancies.

Purpose: In patients with progressive encephalopathy, hypsarrhythmia, and optic atrophy (PEHO) syndrome, the pathophysiology underlying early progressive cerebellar and brainstem degeneration and severe epilepsy is unknown. Because insulin-like growth factor (IGF)-1 has been shown significantly to promote survival of cerebellar neurons, we wanted to see if the IGF system played a role in the pathogenesis of cerebellar atrophy., Methods: We used a sensitive enzyme immunoassay kit for measuring cerebrospinal fluid (CSF) IGF-1 and insulin-like growth-binding protein (IGFBP)-3 in four groups of patients: PEHO syndrome patients (eight), PEHO-like patients (seven), age-matched controls (31), and patients with other types of cerebellar atrophy (11)., Results: Patients with PEHO syndrome and those with other progressive, degenerative cerebellar diseases had lower levels of CSF IGF-1 than the controls with other neurologic diseases. The CSF IGF-1 also allowed us to differentiate the "true" PEHO patients from the "PEHO-like" patients (those with similar clinical symptoms but without the typical neuroophthalmologic or neuroradiologic findings). The concentrations of IGFBP-3 did not significantly differ in any of the patient or control groups studied., Conclusions: CSF IGF-1 levels might be used as a marker of the degeneration of neurons in specific areas.

Objective: To determine whether human vascular endothelial cells produce tumor necrosis factor-alpha (TNF-alpha) after stimulation with proinflammatory cytokines and bacterial lipopolysaccharides (LPS)., Design: Prospective, in vitro repeated-measurements analysis of cellular responses., Setting: Research laboratory in an academic medical center., Subjects: Human umbilical vein endothelial cells (HUVECs)., Interventions: HUVECs were incubated with interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), and LPS, or their different combinations for 2 to 48 hrs., Measurements and Main Results: TNF-alpha was measured by time-resolved immunofluorometric assay. Unstimulated HUVECs did not produce detectable amounts of TNF-alpha, but IFN-gamma, IL-1beta, and LPS when added together induced TNF-alpha production of HUVECs in a time-dependent manner. Immunofluorescent staining confirmed that the TNF-alpha was produced by endothelial cells. IFN-gamma, IL-1beta, or LPS alone did not induce TNF-alpha production, whereas IFN-gamma and IL-1beta in combination were able to induce TNF-alpha production to some extent, and the production could be further increased with LPS. TNF-alpha messenger RNA expression was detected with reverse transcriptase-coupled polymerase chain reaction in stimulated, but not in unstimulated, HUVECs., Conclusions: HUVECs are capable of producing TNF-alpha after proinflammatory cytokine stimulation and may therefore contribute to the increased amount of TNF-alpha found in pathologic states such as septic shock.

Background: To develop an objective measurement system for menstrual blood loss applicable in clinical practice., Methods: One hundred and fifty-six patients were enrolled in a randomized trial of the treatment of menorrhagia in all five gynecology departments of the university teaching hospitals in Finland. Correlation between venous blood hemoglobin concentration (Hb) and absorbance at 564 nm (A564) was investigated in experiments with blood samples. Amount of collected menstrual blood and menstrual diary data were analyzed., Result: Hb concentration and A564 of the blood were linearly correlated (r=0.99). One hundred and fifty-four women (99%) returned used sanitary protection and menstrual diaries. On average, 12% (range 0-57%) of menstrual blood was lost during collection. The median amount of blood recovered from sanitary protection was 89 ml (range 14-724 ml), with 58% (n=90) of the women exceeding 80 ml per cycle. When the spilled blood was taken into account, the corresponding figures were 104 ml (range 15-724 ml) and 66% (n= 101)., Conclusions: The spectrophotometric measurement of venous blood in the conventional alkaline hematin method can be replaced by measurement of Hb concentration. All blood incompletely collected should be recorded. Objective measurement of menstrual blood loss can be applied in routine clinical practice.

Background: The favorable attitude among the public towards prenatal diagnostics in Finland allowed us to start a trial on population basis when screening for Down's syndrome by maternal serum markers and age was introduced., Methods: Screening by maternal serum markers for Down's syndrome was offered to all 17,200 pregnant women in the Helsinki area during the study period of 2.5 years. Screening due to advanced maternal age, 37 years or more, was continued as previously, and 1133 pregnant mothers used this option. Alpha-fetoprotein, human chorionic gonadotrophin, and during the first year also unconjugated estriol were used as markers., Results: The uptake of serum screening was 84%. The proportion of false positive results i.e. risk for Down's syndrome, 1:350 or more at term, was initially 5.7%. After ultrasound scan 4.1% of the mothers remained 'screen positive'. The amniocentesis or chorionic villus sampling uptake was 98.4%. Ten out of eighteen cases of Down's syndrome were detected by maternal serum screening, sensitivity 56%, 95% CI 31-79%. Other chromosomal abnormalities were found in three cases, and there were four cases of mosaicisms confined to the placenta. These were trisomies 16, 7 and 2, and tetraploidy. Elevated serum alpha-fetoprotein was found initially in 0.7% of the cases. One case of congenital nephrosis of the Finnish type and ten other, mainly structural, abnormalities were detected by high AFP., Conclusions: The screening was well received by the mothers. The detection rate of 56% is in the same range as in previous studies. Ultrasound scan before the test would effectively lower the false positive rate caused by incorrect timing.

We here report a reversed-phase HPLC method for the determination of free cortisol in human urine, using methylprednisolone as the internal standard. Before chromatography, samples were extracted with a C18 solid-phase extraction column and the steroids were separated on a LiChrospher 100 C18 column with a mobile phase of methanol/acetonitrile/water (43/3/54 by vol). Linearity, precision, and accuracy of the method were established. The detection limit was 10 pmol of cortisol, and total CVs were < 8%. With various solid-phase extraction columns the recovery of cortisol was 36-97%; recovery of the internal standard was 43-85%. Study of interference by 6 other steroids and metabolites and 24 drugs showed that carbamazepine and digoxin partly overlapped with cortisol, but this interference could be reduced by modification of the mobile phase. The HPLC method was compared with an RIA and an automated immunoassay method. The results obtained by HPLC averaged 40% of the RIA values.

We have developed a 'sandwich'-type time-resolved immunofluorometric assay (IFMA) for tumour necrosis factor alpha (TNF-alpha) using two monoclonal antibodies (mAb) and the streptavidin/biotin (SAB) system. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin coated wells to which we added biotinylated mAb for 3 h. After washing, the serum sample was added and incubated for 2 h followed by washing. Another monoclonal europium-labelled tracer antibody was added and incubated for 1 h, the wells were washed and the fluorescence of Eu measured. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labelled antibody by hydrophobic interaction chromatography was found to be essential to improve sensitivity. With a sample volume of 50 microliters the detection limit was 6 ng l-1 and the analytical range large, i.e. 10,000-fold. The median concentration in serum from healthy subjects was 12 ng l-1 and the reference range < 39 ng l-1. The mean analytical recovery in plasma was 76% and in serum 83%. Separation of serum by gel filtration and assay of TNF-alpha in fractions showed that the assay also measured the high molecular weight (MW) form of TNF-alpha, apparently corresponding to its complex with soluble receptors. Advantages of our SAB-IFMA were high sensitivity and low consumption of mAb. The assay performance of the SAB-IFMA was compared to two commercially available enzyme immunoassays also using the SAB system.

Carbohydrate-deficient transferrin (CDT) has previously been reported to be an excellent marker of male alcoholics. Less is known of its efficiency among women and especially of early-phase alcohol abuse in nonselected populations. The present population-based study examined the diagnostic value of CDT among consecutive women, including 13 teetotallers, 135 social drinkers (mean alcohol consumption 45 +/- 34 g/week), and 57 nonalcoholic heavy drinkers (197 +/- 97 g/week). Sixty-two women with a well-documented history of chronic alcoholism (942 +/- 191 g/week) were also studied, as well as 36 pregnant women used as a reference group. Two weeks of abstinence among 11 alcoholics was followed. The CDT (containing part of isotransferrin with pI = 5.7, 5.8, and 5.9) was separated by anion exchange chromatography and assayed by radioimmunoassay. In the whole material, CDT correlated significantly with alcohol consumption (r = 0.43, p < 0.001) but not with conventional markers (gamma-glutamyltransferase, AST, ALT, and mean corpuscular volume). The CDT values of alcoholics (34 +/- 20 units/liter) were significantly (p < 0.001) higher than those of teetotallers (19 +/- 6 units/liter), social drinkers (20 +/- 6 units/liter), or pregnant women (16 +/- 3 units/liter). Heavy drinkers also had higher values (25 +/- 13 units/liter), but the difference did not reach statistic significance. The specificity of CDT was on the level of conventional markers when the cut-off value was increased from 26 to 29 units/liter. At a specificity of 95%, CDT found 19% of the heavy drinkers and 52% of the alcoholics; the best traditional marker, AST, with a specificity of 97%, found 7% and 56%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We developed a simple, rapid two-step dual-label assay for the noncompetitive determination of alpha-fetoprotein (AFP) and beta subunit of human chorionic gonadotropin (hCG beta) in serum. Monoclonal antibodies to detect AFP and hCG beta were labeled with europium (Eu) and samarium (Sm), respectively. Highly fluorescent chelates were developed by using the Delfia enhancement principle. The detection limits for AFP and hCG beta were approximately 0.02 kIU/L and approximately 0.2 IU/L, respectively. The within-run precision was < 5% over the whole range of AFP (1-500 kIU/L) and hCG beta (1-200 IU/L) concentrations tested. Cross-reaction of intact hCG was < 0.03%. The AFP concentrations determined with the dual-label assay correlated well with those obtained by Delfia AFP single-label kit. The concentrations of hCG beta were in good agreement with recently published data. Storing the serum samples for 24 h or 1 week at room temperature increased the hCG beta concentration by 4% and 26%, respectively. At 35 degrees C this dissociation of hCG increased 30-40-fold. Repeated freezing and thawing had no effect on the hCG beta concentration.

The usefulness of pancreatic secretory trypsin inhibitor (PSTI) as a marker of chronic inflammation was studied in patients with Crohn's disease. Pancreatic secretory trypsin inhibitor was compared with other laboratory tests (C-reactive protein, orosomucoid, and urinary neopterin), for evaluation of disease activity as measured by a clinical scoring system and endoscopical findings. The clinical utility of the tests was compared by four different methods. All tests showed significant differences in laboratory values between inactive and severely active disease. Using earlier established cut-off values PSTI showed the best combination of sensitivity and specificity for differentiation between inactive and severely active disease. When the efficacy of the test was compared by ROC-curve analysis, neopterin and orosomucoid produced the best combination of sensitivity and specificity, but for both assays the earlier established cut-off levels were too high for optimal separation between active and inactive disease. There was a poor agreement between endoscopically and clinically determined disease activity and the laboratory tests correlated better with clinical activity. The present study shows that serum PSTI reflects changes in the clinical activity of Crohn's disease equally well as C-reactive protein, which previously has been found to be an useful index of disease activity.

We have developed a "sandwich"-type time-resolved immunofluorometric assay (IFMA) for fetal hemoglobin (HbF) in hemolysates from adults and newborns, amniotic fluid, and plasma, based on a polyclonal and a monoclonal antibody against human fetal hemoglobin. Microtiter wells are coated with polyclonal capture antibody, and the gamma-chain-specific monoclonal tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted hemolysates are incubated in the microtiter wells first with capture antibody for 1 h and, after washing, for 1 h with tracer antibody. The wells are washed and the fluorescence of europium is measured. The mean analytical recovery is 102% and results by IFMA agreed well with values obtained by high-performance liquid chromatography. The analytical range of IFMA is large and well suited for clinical purposes. The detection limit of the assay is 0.2 microgram/L and the measuring range extends to 500 micrograms/L.

The interpretation of results obtained by immunoassays is affected by many different factors and especially by assay sensitivity and specificity. The introduction of sandwich-type immunometric assays using highly sensitive non-radioactive labels and monoclonal antibodies has greatly improved sensitivity while reducing assay time and simplifying performance. Thus determination of subnormal levels of several hormones has become possible. This approach has also enabled more exact tuning of specificity but at the same time it has introduced the risk of too high selectivity. Hapten assays have also become simpler to perform, but in this case no substantial improvement in assay sensitivity has been achieved.

We describe a simple and rapid immunoturbidimetric end-point assay for measuring pregnancy-specific beta 1-glycoprotein (SP-1) in human serum with the Kone Progress clinical chemistry analyser, using a commercially available antiserum. The absorbance was measured at 340 nm 5 min after addition of antiserum. The assay range was 3-250 mg/l. Over this range the CV for between-day precision was below 6%. Bilirubin up to 160 mumol/l, haemoglobin up to 2 g/l and lipaemia up to 320 mg/l did not interfere. The reference interval in maternal serum at the 15th week of gestation, was 13 to 46 mg/l with a median of 25 mg/l (n = 78) and that of the 16th week was 6 to 60 mg/l with a median of 28 mg/l (n = 78). Comparison of results obtained by two commercially available radio-immunoassays and by the present method shows acceptable correlation. Rapid and reproducible, the immunoturbidimetric method is suitable for use in routine determination of SP-1.

The pharmacokinetics and metabolism of RU 486 were characterized in 17 women who received a single dose of 600 mg of RU 486 for termination of an early unwanted pregnancy. Based on the clinical outcome and serum chorionic gonadotropin values, the subjects were divided into two groups: those who aborted completely (i.e. responders: N = 13) and those who did not respond to RU 486 treatment (i.e. non-responders: N = 4). The serum levels of RU 486, the monodemethylated, didemethylated and hydroxylated metabolites of RU 486 were measured by HPLC preceded by column chromatography. There were no significant differences in the serum levels of RU 486 or its metabolites between the two groups. The serum concentrations of alpha 1-acid glycoprotein, the binding protein for RU 486, were quantitated by immunoturbidimetry. The alpha 1-acid glycoprotein concentrations were similar in responders and non-responders. The metabolism of RU 486 was also studied by fractionating extracts of serum pools of responders and non-responders on thin-layer chromatography, and subsequent RIA analysis of the eluates of the sliced thin-layer chromatography. Spots with similar distribution and percentages of cross-reactivity were found in both groups on the chromatography; the results were also similar to those from a serum pool to which synthetic RU 486 and its three metabolites had been added. Hence it is concluded that failure to abort in response to RU 486 therapy is not associated with altered pharmacokinetics or metabolism of RU 486.

Falsely increased concentrations of the ovarian carcinoma-associated antigen, CA 125, were measured by a monoclonal antibody (MAb)-based double determinant immunoradiometric assay (IRMA) in patients who developed antibodies to mouse immunoglobulins (IgGs) after receiving injections of the same MAb as is used in the CA 125 IRMA. Addition of undiluted mouse serum or purified mouse IgG to the assay mixture failed to eliminate the falsely increased CA 125 concentrations in most of the samples, owing to the presence of anti-idiotype antibody. Because of their anti-idiotypic nature, the human anti-mouse antibodies (HAMAS) had only little effect on other immunometric assays, and this effect could be completely eliminated by addition of mouse IgG. To eliminate the effect of HAMA on the CA 125 assay, we studied the ability of various chromatographic methods to separate the interfering HAMA from CA 125. For measuring HAMA in serum and chromatographic fractions we developed a time-resolved fluoroimmunoassay. Adequate separation of CA 125 and HAMA was achieved by affinity chromatography of patients' sera with solid-phase Protein A, Protein G, cation-exchange chromatography on Mono S, and gel filtration on Superose 6. These results demonstrate that the interference can effectively be removed by rather simple chromatographic procedures.

Tumor-associated trypsin inhibitor (TATI) is a fairly specific serum marker for mucinous ovarian cancer. Very high levels occur in mucinous ovarian cyst fluid, indicating that the elevated levels of TATI in serum are derived from the tumor. However, elevated levels of TATI may also occur in inflammatory disease and after major surgery, suggesting that TATI may also behave as an acute-phase reactant. To further elucidate these questions we have measured the concentration of TATI in urine before and after intracavitary radiotherapy in 13 patients with cervical cancer and 29 patients with endometrial cancer. In 11 patients the concentrations of serum TATI and C-reactive protein (CRP) were also measured. The treatment caused a moderate but significant elevation in the urinary concentration of TATI but affected the serum levels of TATI and CRP only occasionally. The apparent discrepancy between the changes in serum and urine levels of TATI may be explained by the very short (6 min) half-life of TATI in serum. Our results indicate that the mechanisms causing elevation of TATI in urine and of CRP in serum are different. TATI appears to react more rapidly to radiation-induced tissue destruction, whereas CRP is a much more sensitive indicator of bacterial infections.

Urinary dolichol levels of 31 skid-row alcoholics and 49 alcoholics entering a detoxification unit were compared to those of 51 nonalcoholic controls (social drinkers). The mean urinary dolichol content as related to urinary creatinine was significantly (p less than 0.001) higher in the two groups of alcoholics than in the controls. In this material the sensitivity of increased urinary dolichol in the detection of alcoholism was 68% as compared to 44% sensitivity of serum gamma-glutamyltransferase. The percentage of false-positives in the control group was 3.9%. Urinary dolichol is suggested as a potential tool for the detection and follow-up of alcohol abuse.

In this liquid-chromatographic assay for acetylated hemoglobin in human blood, glycated hemoglobins in hemolysates are first removed by affinity chromatography on boronate-agarose columns. Acetylated hemoglobin in the nonretained fraction is determined by cation-exchange chromatography. The absorbance of the effluent is monitored at 415 nm. The mean within-assay CV was 8.5%, the between-assay CV 17%. The mean proportion of acetylated hemoglobin in 20 pregnant, nondiabetic women was 1.9%, and in 17 alcoholics it was 2.7%. Rapid and reproducible, this method is suitable for use in routine determination of acetylated hemoglobin.

The immunoreactivity of human low-molecular-mass kininogen from Cohn plasma fraction IV was investigated after deglycosylations and carbohydrate modifications by radioimmunoassay using the conformation-specific antiserum. Removal of all sialic acids, 44% of amino sugars and 63% of neutral sugars did not alter the immunoreactivity of the protein but the periodate-treated concanavalin A fractions showed strikingly diminished immunoreactivity. A conformational change could account for the observed effect of periodate on the decreased reactivity of the protein in radioimmunoassay. Externally added carbohydrates had no effect on immunoreactivity. The results suggest that the carbohydrate part of kininogen is not involved in the immunoreactivity although it accounts for the observed lectin-binding heterogeneity.

A reversed-phase "high-performance" liquid chromatographic assay for dolichols-18, -19 and -20 in urine is described. Dolichols are extracted from urine by using C18 cartridges and are chromatographed with a mobile phase consisting of 2-propanol/methanol, the effluent being monitored at 210 nm. The useful lower limit of sensitivity for quantification is 4 pmol (5 ng) of each dolichol per 5-microL injection, corresponding to 1.6 nmol (2 ng) per liter of urine. Heneicosaprenol is satisfactory as the internal standard. Peak heights and the amounts of dolichols applied to the column are linearly related from 4 to 110 pmol. Mean analytical recovery was 71%. For three different concentrations the mean within-assay CV was 6.4%, the between-assay CV 11%. The normal reference interval of total dolichols found for healthy adults was 17-101 micrograms/24 h (n = 30) or 1.9-11 micrograms per millimole of creatinine (n = 39). I determined the distribution of the main dolichols in urine and applied the assay also for samples from alcoholics.

Kininogen antigen was purified from human plasma fraction IV by ion exchange chromatography, gel filtration and affinity chromatography with antibody specific immunoadsorbents. The immunologically pure glycoprotein had a mol. wt of approximately 60,000 and only one polypeptide chain by SDS-PAGE. An extensive charge heterogeneity by isoelectric focusing and gel filtration on polyacrylamide agarose could only in part depend on a comparatively high sialic acid content, but may be caused by differences in the carbohydrate structures sustained by lectin-binding heterogeneity on Con A-Sepharose. This antigen shares a dominating determinant with native plasma kininogens shown by complete patterns of identity in immunochemical analyses and with the monospecific antisera developed in rabbits against the heterogeneous components. The similar size, amino acid composition, low histidine content, lack of N-terminal amino acid and antigenic homogeneity fit all the so far known characteristics of the human kininogen heavy chain. Notably the antigenic determinant is resistant to degradation by activated kallikrein. This antigen with unimpaired immunologic activity may be a useful tool for preparation of antiserum for immunochemical determination of human plasma kininogen.

The inhibition of six serine proteinases by a tumour-associated trypsin inhibitor (TATI) was studied using synthetic peptide substrates. Physiological concentrations of TATI inhibited the amidolytic activities of trypsin, plasmin, urokinase and tissue plasminogen activator (tPA). Chymotrypsin, kallikrein and thrombin were also inhibited, but by much higher concentrations of TATI. The ability of TATI to inhibit trypsin, plasmin, urokinase and tPA suggests that it has a role in proteolytic processes in vivo involving these enzymes.

The crystal structure, thermal behaviour, mass spectrum and protonation of 4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole (medetomidine) hydrochloride have been investigated. The title compound crystallizes in both hydrated and anhydrous forms, and their structures have been determined by three-dimensional X-ray structure analysis. The crystals of the anhydrous form are monoclinic and those of the hydrated form (containing one hydrate water molecule) are triclinic with unit-cell dimensions: a = 23.861(9), b = 7.721(4), c = 22.037(9) A, beta = 140.20(4) degrees, Z = 8, and space group C2/c, and a = 7.841(4), b = 8.380(3), c = 12.743(6) A, alpha = 93.66(3), beta = 102.90(3), gamma = 116.85(3) degrees, Z = 2, and space group P1, respectively. Thermal decomposition of the title compound has been interpreted from the TG, DTG and DSC curves with the help of mass spectrometry. Medetomidine hydrochloride monohydrate decomposes in four stages. The first is dehydration at 45-100 degrees C, the second is evaporation of HCl and medetomidine base at 200-320 degrees C, and the third and fourth are decomposition at 340-570 degrees C. The protonation constant is 7.04 in aqueous 0.1 M NaClO4 (25 degrees C).

Native low mol. wt (LMr) kininogen from human plasma and a kinin-free kininogen from Cohn's plasma fraction IV (HC-antigen) were isolated and studied with regard to their immunological reactivity and carbohydrate heterogeneity. Antisera were prepared against the conformational and sequence-dependent determinants of the heavy chain which is the common denominator of plasma kininogens. The molecules were characterized by single radial immunodiffusion and SDS gel electrophoresis. The effect of exoglycosidase treatment was investigated by a radioimmunoassay. It was found that the antigenic combining sites were not influenced by the partial removal of carbohydrates. These results suggest that the determinant structures of LMr kininogen reside in the peptide backbone of the protein. Carbohydrate heterogeneity was shown by different binding to concanavalin A and wheat germ lectin. With concanavalin A 52% of the immunoreactive HC-antigen was reactive compared to 76% of the native LMr kininogen. The weak reactivity (11%) of the HC-antigen towards the wheat germ lectin indicates that the N-acetylglucosamine residues are apparently blocked or missing. Immunoreactive kininogen was in all cases measured by single radial immunodiffusion or an RIA. The molecular nature of the heterogeneity is discussed in relation to the known isoelectric variations of kininogen.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.