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Background: Plasma neutrophil gelatinase-associated lipocalin (pNGAL) has been investigated extensively in acute kidney injury. This study investigated its pathophysiological significance and utility as marker for graft failure and mortality in stable kidney transplant recipients (KTR)., Methods: Baseline pNGAL was measured in 698 KTR (58% male, age 53 ± 13 years, estimated glomerular filtration rate 52.4 ± 20.4 mL/min/1.73 m 2 ) at median 5.4 (interquartile range 1.8-12.0) years after transplantation, enrolled in the prospective TransplantLines Food and Nutrition Biobank and Cohort Study., Results: pNGAL concentrations were higher in males, younger patients, patients with a deceased-donor kidney and higher serum creatinine. Independent of these, pNGAL was positively associated with urinary protein excretion, systemic inflammation parameters and calcineurin inhibitor use. During median follow-up of 5.3 (4.5-6.0) years, death-censored graft failure rates were 3.9%, 7.3% and 25.0% across increasing tertiles of pNGAL (P log-rank  < 0.001). Cox-regression analyses showed no independent associations of pNGAL with mortality, but strong associations with graft failure (hazard ratio, per doubling 4.16; 95% confidence interval 3.03-5.71; P < 0.001), which remained independent of adjustment for confounders. These associations were present only in patients with pre-existent proteinuria and poor kidney function., Conclusions: pNGAL is associated with parameters of kidney graft damage and with graft failure. The latter association is particularly present in KTR with pre-existent poor kidney function and proteinuria. Trial Registration: ClinicalTrials.gov NCT02811835., (© The Author(s) 2021. Published by Oxford University Press on behalf of the ERA.)

Background: Neutrophil gelatinase-associated lipocalin (NGAL), a novel biomarker of acute kidney injury, might play a role in the development of atherosclerotic cardiovascular disease (CVD). We aimed to assess the association of circulating NGAL with CVD risk., Materials and Methods: Plasma NGAL concentrations were measured at baseline in 5275 participants in the PREVEND prospective study. Hazard ratios (95% confidence intervals [CI]) for CVD were estimated., Results: After a median follow-up of 8.3 years, 338 participants developed first CVD events. Plasma NGAL was weakly to moderately correlated with several CVD risk markers. There was a non-linear relationship between NGAL and CVD risk. In analyses adjusted for established risk factors, the hazard ratio (95% CI) for CVD in a comparison of the top quartile versus bottom quartiles 1-2 of NGAL values was 1.35 (1.05-1.75; P = 0.022), which was abrogated after additional adjustment for other potential confounders (mainly attributed to high sensitivity C-reactive protein) 1.20 (0.92-1.57; P = 0.176). The association was considerably attenuated following further adjustment for renal function 1.05 (0.79-1.40; P = 0.745). The association between NGAL and CVD risk did not vary importantly in relevant clinical subgroups., Conclusion: Evidence suggests a non-linear association between NGAL and CVD risk, which is dependent on inflammation and renal function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Aims: We aimed to assess the association of circulating calprotectin, an inflammation-associated protein, with cardiovascular disease (CVD) risk and determine whether it improves risk prediction., Methods: Plasma calprotectin measurements were made at baseline in 5290 participants in the PREVEND prospective study. Hazard ratios (95% confidence intervals [CI]) for CVD were calculated., Results: After a median follow-up of 8.3 years, 339 first CVD events were recorded. Calprotectin concentration was correlated with several conventional risk factors as well as with high-sensitivity C-reactive protein (hsCRP) (r = 0.42). Calprotectin was log-linearly associated with CVD risk. The risk for CVD adjusted for conventional cardiovascular risk factors was 1.26 (95% CI, 1.13-1.41) per 1 standard deviation higher baseline log e calprotectin, and was 1.24 (95% CI, 1.11-1.39) following further adjustment for triglycerides, body mass index, and other potential confounders. The association remained present after further adjustment for hsCRP 1.15 (95% CI, 1.02-1.30). Comparing extreme quartiles of plasma calprotectin levels, the corresponding adjusted HRs for CVD were 1.96 (1.37-2.82), 1.89 (1.31-2.72), and 1.56 (1.07-2.29). The association of calprotectin with CVD risk did not vary importantly in several relevant clinical subgroups. Adding calprotectin to the Framingham CVD Risk Score was associated with a C-index change (0.0016; p=0.42) difference in -2 log likelihood (p=0.038), IDI (0.0080; p < 0.001), and NRI (4.03%; p=0.024)., Conclusions: There is a log-linear association of calprotectin concentration with risk of CVD, which may be partly dependent on hsCRP. Adding calprotectin to conventional risk factors improves CVD risk assessment using measures of reclassification and -2 log likelihood., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin., Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >10(10) L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin., Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8-9%, respectively. The working range (CV <20%) was 5-370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93-108%. A 95% reference interval of 24-157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20-80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r(2) = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r(2) = 0.23) with total cobalamin or holo-haptocorrin., Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

Primary biliary cirrhosis (PBC) is one of the few non-alcohol induced liver pathologies which causes false positive results in the evaluation of carbohydrate-deficient transferrin (CDT) for the diagnosis of alcohol misuse. This phenomenon has only been observed when using the CDTect assay (Pharmacia & Upjohn, Uppsala, Sweden). In this study, we evaluated CDT in female PBC patients (n = 14) by a new CDT procedure, the %CDT turbidimetric immunoassay (TIA, Axis Biochemicals, Oslo, Norway) using the isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD, Specialty Laboratories, Santa Monica, CA, USA) procedure as the gold standard. One of the PBC patients tested CDT+ by IEF/IB/LD (cut-off >9 densitometry units, DU) and %CDT TIA (cut off >6%); one patient tested at the cut-off point of the IEF/IB/LD and another one tested at the cut-off point of the %CDT TIA. Thus, unlike CDTect, the %CDT TIA is a procedure that produces few false positives in PBC.

We present a new filter assay for the determination of glycohemoglobin as a unique application of the boronic acid affinity principle. With the use of a water-soluble blue-colored boronic acid derivative and a specific precipitation method for hemoglobin, total hemoglobin including bound boronic acid is precipitated and collected on a filter strip before quantification. Hemoglobin and boronic acid are quantified by a dual-wave-length reflectometric measurement, and the result is reported directly as percent glycohemoglobin. The test is simple, quick, and designed as a doctors' office test for the monitoring and management of diabetes. The imprecision of the assay is < 4% over the range 3-18% Hb A1c, and the method is linear up to at least 20% Hb A1c. Comparisons with four well-established glycohemoglobin methods yielded correlation coefficients ranging from 0.94 to 0.99, with slopes from 0.94 to 1.01.

Water soluble dye-phenylboronic acid conjugates (dye-PBAs) possessing strong absorption of visible light are introduced as new reagents for the determination of glycohemoglobin. Their functionality and prospective use are demonstrated in a semi-homogenous glycohemoglobin assay. The assay is based on cis-diol esterification of dye-PBA to glycohemoglobin followed by selective precipitation of hemoglobin from solution, co-precipitating bound dye-PBA. Quantification of the molar "dye-PBA/Hb"-ratio in redissolved precipitates using either absorption or fluorescence spectroscopy, reflects the glycation level of the blood samples used. Future development of the assay principle is illustrated in a filter based assay, collecting the precipitated hemoglobin on a filter followed by reflectometric readings directly on the precipitate. The significance of this work lies first, in the demonstration of a new principle for the determination of glycohemoglobin, and second, as an illustration of the prospective use of water soluble, signal-forming non-immobilised boronic acids in the determination of cis-diol containing analytes.

Carbohydrate-deficient transferrin (CDT) may now be the most valuable biological marker for diagnosis of alcohol abuse. We compared the diagnostic performance of two new CDT tests, Axis %CDT turbidimetric immunoassay (TIA) and Axis %CDT HPLC, against Specialty Laboratories' isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD). Both Axis tests include one-half the concentration of trisialotransferrin isoforms in their CDT quantitation schemes. Considering an alcohol abuse prevalence of 7%, Axis %CDT TIA shows a sensitivity of 87% at 98% specificity and a positive predictive value (PPV) of 0.75; %CDT HPLC shows a sensitivity of 87% at 100% specificity for a PPV of 1, and the IEF/IB/LD shows 81% sensitivity at 94% specificity for a PPV of 0.5. All three CDT tests show the same negative predictive value (0.98). Both Axis procedures perform better than IEF/IB/LD in the diagnosis of alcohol abuse; %CDT TIA is available in several semiautomated, cost-effective formats.

Recent studies have demonstrated that the diagnostic performance of CDT measurements is influenced by variations in total transferrin concentration. Improved specificity is obtained with relative values, either %CDT or units CDT per total transferrin concentration. Whether relative or absolute values are used, confirmatory methods by HPLC or IEF should be available, and definition of the CDT values and correlation to HPLC or IEF should be given.

The interaction of human hemoglobin with several organic solvents and metal cation has been studied in order to obtain selective precipitation of hemoglobin from solution. Alcohols, and preferably the mixture ethanol: butanol added to a final concentration of 8% (v/v) 1-butanol was found to be superior in this respect, giving close to selective precipitation of hemoglobin from whole blood lysates. An equally specific precipitation was achieved by using zinc-chloride in 10-15 molar excess to hemoglobin. Contrary to organic solvents, complex formation with Zn2+ resulted in a reversible precipitation enabling renaturation using strong chelating agents. Specificity of the hemoglobin-precipitating agents was verified by chromatographic and electrophoretic studies. Applications of the presented methods in analytical chemistry and in the isolation and purification of blood proteins are discussed.

CDT (carbohydrate-deficient transferrin) has been identified as a specific marker for chronically elevated alcohol consumption. We investigated the sensitivity and accuracy of using relative concentrations of different isotransferrins in serum for diagnosis of chronically elevated alcohol consumption. The different transferrin variants (isoforms) were quantified by HPLC. Including the trisialo-transferrin fraction into the definition of %CDT resulted in an increased accuracy in the detection of chronically elevated alcohol intake in a study among 17 heavy drinkers, 25 healthy individuals with moderate alcohol consumption and nine total abstainers. The results also suggest that desialylation of transferrin is a gradually continuing process, rather than one leading to a single end-result separating asialo-, mono- and disialo-transferrins from trisialo-, tetrasialo-, pentasialo- and higher sialo-transferrins.

We investigated the usefulness of the laboratory marker of alcohol consumption carbohydrate-deficient transferrin (CDT) in 101 consecutively admitted patients in a surgical and internal medical ward of a hospital in a rural wine-growing area. Four major aspects were considered: the influence of liver disease, the method of expression of CDT values (relative % vs absolute units/1), level and pattern of alcohol consumption and comparison with y-glutamyl transferase (GGT). The results show that %CDT is a more valuable discriminating marker of high alcohol consumption than absolute CDT values and its usefulness in this respect is independent of changes in serum total transferrin levels, as in liver disease. Sensitivity and specificity of % CDT were 70 and 98% respectively, compared with 65 and 83% respectively for GGT.

This paper reports the results of a 3-week drinking experiment in 51 healthy male subjects, examining the value of %CDT (carbohydrate-deficient transferrin) in the context of different levels of alcohol intake. All healthy persons were urine-tested drug-free and underwent daily breath alcohol tests for the 7 days preceding, and during the whole 3 weeks of, the experiment. Subjects were divided into five groups, consuming different amounts of alcohol daily over a 3-h period in the presence of the investigators. The five groups consisted of 10, 9, 10, 16 and 6 subjects respectively and consumed a daily dose of ethanol of 20, 40, 60, 80 and 80 g respectively for 3 weeks. No significant changes in %CDT were detected in most subjects, even in the 80 g alcohol-consuming groups. The results suggest that CDT is not sensitive for the detection of short-term heavy drinking by healthy subjects.

We have studied the interaction of Concanavalin A and Lentil Lectin with glycohaemoglobin by a nephelometric lectin-glycogen/dextran precipitation system and monitored the inhibitory effect of glycohaemoglobin on the precipitation. Although inhibitory effects were clearly demonstrated using simple sugars and transferrin, no effect was observed by glycohaemoglobin in relevant concentrations. This is compared to affinity chromatography, binding studies using gel filtration and electrophoresis, and affinity studies using Concanavalin A immobilised on magnetisable polymer particles. Lack of interaction between glycohaemoglobin and lectins is discussed in view of steric constraints and reduced availability of the glycated residues and the stereochemical form of the glycated 1-amino-1-deoxy-fructosyl residues in glycohaemoglobin.

Both disruption of the native protein structure and oxidation of iron in the haem/iron(II)-proto-porphyrin-IX residues were observed using the Iodo-gen (1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycouril) method in 125I-labelling of haemoglobin. The reactions taking place affect the native structure of haemoglobin and result in a more acidic molecule. The detrimental effects were unaffected by the presence of iodine. Electrophoretic studies demonstrate that 125I-labelling of haemoglobin using the Bolton-Hunter reagent is the method of choice in order to preserve the native protein.

Different methods for covalent linkage of phenylboronic acid (PBA) to structural proteins and enzymes are presented. Protein-PBA conjugates, free in solution or immobilised on magnetizable polymer particles, were tested for their binding of D-sorbitol, D-mannose and glycohemoglobin (GHb). Similarly, alkaline phosphatase-PBA conjugates were used in an attempted enzyme-linked sorbent assay for the detection of GHb. Affinity chromatography on immobilised D-mannose and gel chromatographic studies of protein-PBA complexes with [14C]sorbitol, clearly illustrated a low affinity of the interaction studied. Glycated hemoglobin could not be detected using the enzyme-linked sorbent assay approach. However, GHb was found to be specifically retained on columns filled with protein-PBA-coated particles as affinity matrix, enabling the glycation level of blood samples to be determined.

The possibility of obtaining useful scintigrams of secondary lymphoid organs after infusion of syngeneic lymphocytes labelled with technetium-99m (99mTc) was explored in a rat model. Thoracic duct lymphocyte (TDL) accumulation in various organs was measured with both 99mTc and 51Cr labelled cells, the latter processed with a method that has been shown not to damage lymphocytes. 99mTc labelled TDL did not localize properly in the lymph nodes and spleen. We could not visualize lymph nodes in scintigrams, neither could we demonstrate any difference between normal and hyperplastic spleens. Our conclusion is that radiation from the 99mTc label readily influences lymphocyte migration so that useful scintigraphy in rats and other small experimental animals becomes impossible. This was supported by results from culture experiments with 99mTc labelled, radiosensitive mouse haemopoietic progenitor cells. Theoretical considerations, including the calculations of lymphocyte self-irradiation and signal/noise ratios during scintigraphy of rat tissues, supported our conclusion that scintigraphy in small animals, to disclose the physiological migration of lymphocytes, may be impossible with the present sensitivity of gamma cameras.

A new method for 99mTc-labeling of granulocytes for clinical routine use has been developed. The labeling is simple to perform by means of a kit of radiopharmaceutical quality, utilizing dihydroxy-benzoic acid. Pretinning techniques are avoided. The technique has been applied clinically in 15 patients with indications of intra-abdominal abscess. In six patients, [99mTc]granulocyte scintigraphy at 3 hr and/or 24 hr after i.v. administration, correctly depicted the abscess, as verified by subsequent surgery. In the remaining patients, who were negative at surgery or recovered without operation, all scans were negative.

The concentrated hydrochloric acid/vacuum evaporation method has been applied to form 99mTc-plasmin complexes free from radiocolloids for medical scintigraphy. The yield and radiochemical purity were insufficient for medical application when no antioxidant was present during the complex formation. With gentisic acid present the yield was increased by a factory of 2-3, and high radiochemical purity was obtained. No preparative chromatography was necessary. Presence of ascorbic acid inhibited the 99mTc-plasmin formation.

A method for the formation of 99mTc-human immunoglobulin G(99mTc-IgG) complexes free from radiocolloids is described. A concentrated hydrochloric acid reduction/vacuum evaporation/gentisic acid method was applied. Although no preparative chromatography was performed, no sign of 99mTc-radiocolloids was observed in the chemical quality controls of the 99mTc-IgG formation. High radiochemical purities of 99mTc-IgG were obtained either by precipitation in 32 vol.% ethanol in water at -10 degrees C, or by removal of other 99mTc-complexes by simply adding a suspension of sterilized Dowex 1 in gentisic acid solution. Besides gel chromatography and thin layer chromatography, the 99mTc-IgG formation was quality controlled by radioimmunoelectrophoresis in agarose gel with rabbit anti-human IgG-antibodies present in the gel. The 99mTc-IgG complexes were complex bound to the rabbit anti-human IgG antibodies, and precipitated as rockets in the gel. These rockets could be scintigraphed and their radioactivities could be quantified, thereby a quantitatively radio-chemical quality control based on the antigenicities of the 99mTc-IgG complexes was obtained. The 99mTc-IgG complexes were highly stable with no sign of dissociation even when the radioimmunoelectrophoresis was performed for 24 h. Improvements of the radioimmunoelectrophoretic system for the quality control of 99mTc-complexes with specific purified antibodies, are proposed.

The whole body distribution of radioactivity as a function of time after infusion of 99mTc labelled autologous granulocytes was measured in three volunteers by means of a scanning bed, a scintillation camera and a minicomputer. Labelling was performed with a bisalt method without pretinning. There was a considerable initial lung sequestration (22%-31%) of the injected activity, which disappeared with an effective half life of 42 min. One h after infusion the activity was found mainly in the liver (41%), spleen (8%), lungs (9%) and kidneys (5%). Urine excretion amounted to 30% during the first 32 h after infusion. An injected activity of 100 MBq caused a radiation dose of 4.4 m Gy to the liver, 6.3 m Gy to the spleen, 3.7 m Gy to the kidneys, and 0.2 m Gy and 0.1 m Gy to the ovaries and testes respectively. The labelling procedure and the subsequent decay within the granulocytes gave them an absorbed radiation dose of 1.8 Gy after 25 min (i.e., at completion of the infusion) and 8.4 Gy after 4 h (i.e., the normal imaging time). In vitro tests revealed no signs of radiation damage to the cells.

Large fractions of immunoglobulin G (IgG) from mammalian species show high affinities for protein A isolated from staphylococcus aureus. The radiochemical purity of 99mTc-labelled IgG was determined by means of protein A, covalently bound to sepharose, by a column radiochromatographic technique and by a simpler and more rapid technique in vials. Different labelling methods produced different radiochemical purities. Limitations and applications of the testing systems are discussed.

99mTc-complexes with the polypeptide hormone secretin in very low concentration were formed by the concentrated hydrochloric acid/vacuum evaporation/gentisic acid method. The 99mTc-secretin was characterized by a modified ampholyte radiochromatographic procedure, in addition to thin layer chromatography, gel chromatography and paper electrophoresis. High radiochemical purity and specific radioactivity were obtained. In vivo distribution studies were performed, and the conditions necessary for application of [99mTc]polypeptides as scintigraphic agents are discussed.

99mTc -platelets and 99mTc -leucocytes have been formed at neutral pH in presence of gentistic acid using 99mTc pretreated with concentrated hydrochloric acid and vacuum evaporation. High radiochemical purities and specific radioactivities were obtained. The complexes between 99mTc and the blood cells were found remarkably stable, and further studies for nuclear medicine applications are recommended.

The oxidation state of technetium-99m, reduced by concentrated hydrochloric acid and evaporation until dryness, was determined by radiochromatography. The dry deposits of reduced 99mTc were allowed to react with DTPA and MDP with and without antioxidants present. The formation of 99mTc(IV)-DTPA and 99mTc(IV)-MDP complexes and the influence of the antioxidants gentisic and ascorbic acids were studied by radiochromatography and by biodistributional studies in rabbits. 99mTc(IV)-MDP complexes thus formed were demonstrated to have the same biodistribution in rabbits as 99mTc-MDP complexes formed by a conventional stannous reduction kit technique.

To establish a method for radiochemical quality control of [99mTc]plasmin based on the known affinity of plasmin for lysine residues, human [113Sn]plasmin and [99mTc]plasmin formed by different methods were analyzed in an affinityradiochromatographic system of lysine coupled to CNBr-activated sepharosis. From the observed immobilizations of the radioactivity when the plasmin was bound to the lysine-sepharosis, radiochemical purities of radiolabelled plasmin could be calculated. The effects of presence of Sn(II) and tartaric and gentisic acid and the remobilizations induced by 6-aminohexanoic acid were studied.