Characterization of soluble folate receptors (folate binding proteins) in humans. Biological roles and clinical potentials in infection and malignancy.

This review surveys soluble Folate Receptors (FOLRs) in humans. FOLR1 and FOLR2 are equipped with cellular glycosylphosphatidylinositol (GPI) anchors. FOLR1 is secreted from epithelia with or without a micelle-encapsulated GPI-anchor into milk and other body fluids/secretions, e.g. semen where its interaction with spermatozoa indicates a role in male fertility. FOLR1 and FOLR2 serve as serum biomarkers of various diseases. FOLR3 possesses no GPI-anchor and originates from secretory granules of neutrophil granulocytes; its concentration in serum correlates to the FOLR3 content in leukocytes and rises with increased leukocyte counts (infection, malignancy and pregnancy). FOLR3 exerts anti-microbial and anti-tumor effects by depriving bacteria and tumor cells of natural folates. Megalin receptors mediate reabsorption of ultrafiltered folate-bound FOLR into cells of proximal kidney tubules and of folate-bound FOLR uptake in growing embryos. Megalin receptors overexpressed in malignant tumors could be suitable therapeutic targets for folate-conjugated cytotoxic agents utilizing soluble FOLRs as vectors.

Plasma creatinine medians from patients partitioned by gender and age used as a tool for assessment of analytical stability at different concentrations.

Background Monthly medians of patient results are useful in assessment of analytical quality in medical laboratories. Separate medians by gender makes it possible to generate two independent estimates of contemporaneous errors. However, for plasma creatinine, reference intervals (RIs) are different by gender and also higher over 70 years of age. Methods Daily, weekly and monthly patient medians were calculated from the raw data of plasma creatinine concentrations for males between 18 and 70 years, males >70 years, females between 18 and 70 years and females >70 years. Results The medians of the four groups were all closely associated, with similar patterns. The mean of percentage bias from each group defined the best estimate of bias. The maximum half-range (%) of the bias evaluations provided an estimate of the uncertainty comparable to the analytical performance specifications: thus, bias estimates could be classified as optimum, desirable or minimum quality. Conclusions Medians by gender and age are useful in assessment of analytical stability for plasma creatinine concentration ranging from 60 to 90 µmol/L. The daily medians are valuable in rapid detection of large systematic errors, the weekly medians in detecting minor systematic errors and monthly medians in assessment of long-term analytical stability.

Gender-partitioned patient medians of serum albumin requested by general practitioners for the assessment of analytical stability.

BACKGROUND: Recently, the use of separate gender-partitioned patient medians of serum sodium has revealed potential for monitoring analytical stability within the optimum analytical performance specifications for laboratory medicine. The serum albumin concentration depends on whether a patient is sitting or recumbent during phlebotomy. We therefore investigated only examinations requested by general practitioners (GPs) to provide data from sitting patients. METHODS: Weekly and monthly patient medians of serum albumin requested by GP for both male and female patients were calculated from the raw data obtained from three analysers in the hospital laboratory on examination of samples from those >18 years. The half-range of medians were applied as an estimate of the maximum bias. Further, the ratios between the two medians were calculated (females/males). RESULTS: The medians for male and female patients were closely related despite considerable variation due to the current analytical variation. This relationship was confirmed by the calculated half-range for the monthly ratio between the genders of 0.44%, which surpasses the optimum analytical performance specification for bias of serum albumin (0.72%). The weekly ratio had a half-range of 1.83%, which surpasses the minimum analytical performance specifications of 2.15%. CONCLUSIONS: Monthly gender-partitioned patient medians of serum albumin are useful for monitoring of long-term analytical stability, where the gender medians are two independent estimates of changes in (delta) bias: only results requested by GP are of value in this application to ensure that all patients are sitting during phlebotomy.

Separate patient serum sodium medians from males and females provide independent information on analytical bias.

BACKGROUND: During monitoring of monthly medians of results from patients undertaken to assess analytical stability in routine laboratory performance, the medians for serum sodium for male and female patients were found to be significantly related. METHODS: Daily, weekly and monthly patient medians of serum sodium for both male and female patients were calculated from results obtained on samples from the population >18 years on three analysers in the hospital laboratory. The half-range of medians was applied as an estimate of the maximum bias. Further, the ratios between the two medians were calculated. RESULTS: The medians of both genders demonstrated dispersions over time, but they were closely connected in like patterns, which were confirmed by the half-range of the ratios of medians for males and females that varied from 0.36% for daily, 0.14% for weekly and 0.036% for monthly ratios over all instruments. CONCLUSIONS: The tight relationship between the gender medians for serum sodium is only possible when raw laboratory data are used for calculation. The two patient medians can be used to confirm both and are useful as independent estimates of analytical bias during constant calibration periods. In contrast to the gender combined median, the estimate of analytical bias can be confirmed further by calculation of the ratios of medians for males and females.

Elevated prostate specific antigen and reduced 10-year survival among a cohort of Danish men consecutively referred from primary care to an urological department during 2005-2006.

It remains unclear whether total prostate specific antigen (tPSA) or complex PSA (cPSA) has the best diagnostic performance. Additionally, the utility of percentage free PSA (%fPSA) is still debated. Our objectives were to compare the diagnostic performances of tPSA, cPSA, and %fPSA among patients referred from GP to an Urological Specialist and to investigate prognostic factors and survival in the cohort. A total of 1261 consecutive male patients without previously known prostate cancer (PCa) were referred to the same Department of Urology during June 2005 to August 2006. Some 299 patients were diagnosed with PCa and 962 patients were found without PCa. Among the PCa patients, the median age, tPSA, cPSA, and %fPSA levels were 70.8 years, 13.4 µg/L, 10.8 µg/L, and 12.6%. For patients without PCa the results were 67.5 years, 2.5 µg/L, 1.9 µg/L, and 24.9%. The sensitivity, specificity, PVpos, PVneg, and efficiency of tPSA and cPSA were overlapping (p > .05). In the tPSA interval >4 µg/L - ≤20 µg/L, %fPSA excluded PCa with a PVneg of 72.4%; 38.5% of PCa patients had a tPSA concentration >20 µg/L at the time of referral and these patients had a reduced 10-year survival as compared to patients with tPSA concentrations ≤20 µg/L. In conclusion, tPSA and cPSA showed similar diagnostic performances. %fPSA provided additional diagnostic information at tPSA concentrations >4 µg - ≤20 µg/L. The high percentage of patients with tPSA concentrations >20 µg/L indicate delayed use of tPSA resulting in advanced disease at presentation and reduced patient survival.

Median of patient results as a tool for assessment of analytical stability.

BACKGROUND: In spite of the well-established external quality assessment and proficiency testing surveys of analytical quality performance in laboratory medicine, a simple tool to monitor the long-term analytical stability as a supplement to the internal control procedures is often needed. METHOD: Patient data from daily internal control schemes was used for monthly appraisal of the analytical stability. This was accomplished by using the monthly medians of patient results to disclose deviations from analytical stability, and by comparing divergences with the quality specifications for allowable analytical bias based on biological variation. RESULTS: Seventy five percent of the twenty analytes achieved on two COBASs INTEGRA 800 instruments performed in accordance with the optimum and with the desirable specifications for bias. DISCUSSION: Patient results applied in analytical quality performance control procedures are the most reliable sources of material as they represent the genuine substance of the measurements and therefore circumvent the problems associated with non-commutable materials in external assessment. CONCLUSION: Patient medians in the monthly monitoring of analytical stability in laboratory medicine are an inexpensive, simple and reliable tool to monitor the steadiness of the analytical practice.

A chemometric analysis of ligand-induced changes in intrinsic fluorescence of folate binding protein indicates a link between altered conformational structure and physico-chemical characteristics.

Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate)-induced changes in the fluorescence landscape of FBP. Fluorescence excitation and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important finding was the occurrence of two maximum intensity emission wavelengths of tryptophan, 350 nm (component one) and 330 nm (component two). In contrast to the first component, the score of the short wavelength component increased with increasing ligation of FBP. Since the emission wavelengths of indole groups in tryptophan shorten with increasing distance from the solvent surface of proteins, an increasing number of the 11 tryptophan residues seem to reorientate from the solvent surface to the interior of FBP with increasing ligation. The sharp decrease in hydrophobicity at pI=7-8 following binding of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition with radiofolate. This could suggest a rapid and firm complexation of folate to FBP, blocking access of competing ligands.

Stopped-flow kinetic studies of the interaction of bovine folate binding protein (FBP) and folate.

The kinetics of the interaction of bovine folate binding protein and folate at pH 7.4 and 5.0 were followed by measuring the changes of the intrinsic protein fluorescence intensity using the stopped-flow technique, which enables the study of reactions from the millisecond time-range. Our results immediately reject a simple one-step binding model, which requires a linear dependence of the observed rate constant on the concentration of the ligand. Thus, we are able to conclude that at pH 5.0 the interaction occurs in two steps and at pH 7.4 in three steps. Changes of fluorescence spectra at equilibrium were used to estimate the overall binding constants. Comparative studies on the binding of folate to human albumin are also reported.

Application of near-infrared and Fourier transform infrared spectroscopy in the characterization of ligand-induced conformation changes in folate binding protein purified from bovine milk: influence of buffer type and pH.

Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a beta-sheet to alpha-helix transition upon ligation in acetate and phosphate buffers, and the formation of intermolecular beta-sheet was indicated at pH 5.0, in agreement with a dimerization of FBP taking place at this pH. The ligand-induced changes in the 2100-2300 nm NIR region were significant for FBP in acetate and phosphate buffers of pH 5.0, and the variations were interpreted as secondary structure changes, based on previous assignments of secondary structures to the combination bands in the NIR region. In the case of acetate buffer, variations in the amide combination bands agreed with the amide I analysis, but for the other buffer types some discrepancies were found and explained by side-chain contributions to the NIR, which could reflect the tertiary and quaternary structure differences. NIR spectra of FBP at pH 7.4 and 5.0 revealed contradictory effects on the side chains, reflecting different polymerization events at the two pH values, whereas the amide I region indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT-IR, the tertiary and quaternary structure changes are reflected in the NIR spectra, although the general influence of the latter changes on the NIR spectra remains to be confirmed.

A minor fraction of a high-affinity folate binding protein from the epididymis is associated with membranous vesicles and spermatozoa in human semen.

A glycolipid linked high-affinity folate binding protein (FBP) is present in human semen at a concentration of 1-2 nmol/L. The association between FBP and seminal components as well as the cellular source of FBP was analysed. Immunoblotting of human seminal plasma, with and without prostasomes, prostasomal fractions and spermatozoa with antibodies against human FBP revealed a single distinct band similar to that observed with purified human FBP. Flow cytometry identified FBP on the surface of ejaculated spermatozoa. Immunohistochemistry showed positive immunostaining of epididymal epithelium, vas deferens and ejaculated spermatozoa, whereas the prostate gland, seminal vesicles, testicular spermatozoa and seminiferous tubules of testis stained negatively. Electron microscopy immunocytochemistry with antibodies against rat FBP showed labelling located to luminal microvilli and intracellular vesicles of rat epididymal epithelial cells and the surface of spermatozoa in the epididymal duct. High-affinity binding of picomolar amounts of tritiated folate to fractions of human prostasomes or prostasome-like vesicles was completely depressed by excess amounts of unlabelled folate. The study indicates that FBP is secreted from the epithelia of epididymis and vas deferens, and that a small fraction of FBP is associated with prostasome-like vesicles which adhere to spermatozoa in the epididymal duct. FBP could have a bacteriostatic function by depriving folate-requiring bacteria of folate and/or ascertain a normal DNA replication subsequent to fertilization by vectorial transfer of folate to the inner compartment of the spermatozoa.

Ligand binding and polymerization characteristics of human milk folate binding protein depend on concentration of purified protein and presence of amphiphatic substances.

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.

Characterization of a high affinity folate binding protein in porcine serum: ionic charge, concentration-dependent polymerization and ligand binding mechanism.

The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-affinity type with upward convex Scatchard plots and Hill coefficients > 1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/l. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing hetecrodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.

A combination of cation exchange and ligand-affinity chromatography for purification of two molecular species of the folate binding protein in human milk, one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge.

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5-6 was a cleavage product of a 100 kDa protein eluted at pH 3-4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7-9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.

Ligand binding characteristics of two molecular forms, one equipped with a hydrophobic glycosyl phosphatidylinositol tail, of the folate binding protein purified from human milk.

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [3H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.

Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol, phospholipids, and synthetic detergents.

Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.