Isoelectrofocusing Research Articles

Infrequent patterns in cerebrospinal fluid isofocusing test: Clinical significance and contribution of IgG index and Reiber diagram to their interpretation

Cerebrospinal fluid (CSF) isoelectrofocusing (IEF) is considered as the gold standard for detecting an intrathecal synthesis of IgG, which is a hallmark of multiple sclerosis (MS). This corresponds to the presence of CSF-restricted IgG oligoclonal bands (OCB) (typically type 2 pattern). Moreover, this technique can also detect a systemic immune reaction with passive transfer of IgG (type 3 and 4 patterns) for which the clinical relevance is less understood. The aim of our study was to determine the frequency and disease associations of IEF type 3 and 4 patterns and to investigate the potential usefulness of including quantitative data (IgG index and Reiber Diagram) in interpreting such IEF profiles.Among 544 patients who underwent CSF IEF (Hydragel CSF isofocusing kit, Sebia®, France) in our Laboratory during a six-year-period, those who presented type 3 or 4 patterns were selected. Clinical data and results of other immunological tests were analyzed for 27 patients followed in the Neurological Department.Frequencies of type 3 and type 4 patterns were relatively low (2.3 % and 3.4 % respectively). Among patients with type 3 pattern included in our study (n = 10), 5 were diagnosed with MS. For the 5 other patients, the diagnosis was a clinically isolated syndrome (CIS) (2 cases), a probable auto-immune encephalitis (2 cases) and a possible genetic neurodegenerative disease (1 case). MS patients had an IgG index >0.7 and fell into area 4 of Reiber diagram while non-MS patients had an IgG index <0.7 and fell into area 1, except the last case. Regarding type 4 pattern (n = 17), the diagnoses were as follows: MS (3), CIS (4), Neuromyelitis optica spectrum disorders with positive anti-AQP4 antibodies (3) and anti-NMDAR autoimmune encephalitis (1). The remaining cases had central nervous system impairment related to vascular, metabolic or tumoral etiologies (3) or peripheral nervous system impairment (3). In this group (type 4 pattern), IgG index was <0.7 in 15/17 cases. Interpretation using Reiber diagram showed an abnormal blood-brain barrier for 8/17 patients.Type 3 and 4 IEF patterns are infrequently observed in routine neurology practice. It is important for the diagnostic laboratory professional as well as for the neurologist to understand their clinical relevance. Our findings highlight the contribution of quantitative evaluation of CSF (IgG index, Reiber diagram) for the interpretation of such situations. Despite the small size of our study population, our results emphasize the importance of reporting the exact type of IEF pattern and not only the positivity or not of OCB.

Analysis of the immunomodulatory activity of excreted and secreted products from Haemonchus placei transition infective larvae (xL3)

The excretory/secretory (E/S) products released by infective transitory larvae (xL3) of Haemonchus placei have an important biological function in stimulating immune mechanisms during the invasive process. Our objective was to analyse the modulatory activity of 15 and 70 kDa E/S products from H. placei xL3. Both E/S products were collected from xL3in vitro cultures at 24 and 72 h. Proteins were confirmed by SDS-PAGE, and the corresponding spots were elicited by gel isoelectrofocusing (IEF) and characterised by mass spectrometry. Additionally, flow cytometry of CD4+/γδ+ T cells and immune gene expression were performed by proliferation assays using each E/S product to stimulate lymphocyte and peripheral blood mononuclear cells (PBMCs) from non-infected calves. The IEF results displayed two spots of 7.0 and 5.7 pI for the 15 and 70 kDa products, respectively. Additionally, 29 and 17 peptides from the 15 and 70 kDa E/S products, respectively, were identified with the hypothetical neurotransmitter and enzymatic functions necessary for larval development. The relative expression displayed upregulation of IL4, 5, 6, 8, 10, 13, IFNγ, and FCεR1A genes (from 2.0- to 17.6-fold, p < 0.05) stimulated by the 15 and 70 kDa proteins, indicating specific genes against haemonchosis. Although the percentage of median florescence intensity (MFI%) of CD4+/γδ+ T cells did not change for both E/S products compared to the negative control and concanavalin-A stimulated cells as the positive control (p > 0.05), the 15-kDa protein reduced the levels of both T cells, and the 70-kDa proteins increased the γδ+ cells slightly. Additionally, there was increased PBMCs proliferation by the 70 kDa proteins (p < 0.05), denoting the biological role of other immune cells. The 15 and 70 kDa protein E/S products from H. placei xL3 showed modulation of the immune response, and although more studies are required, they indicate important functions in the host/parasite interaction.

New forms of home dust mite allergoid

Aim. The purpose of this study is to create the technology of preparation of the polymeric allergoid received by chemical modification of Dermatophagoides farinae extract by a formaldehyde and a monomeric allergoid of Dermatophagoides pteronyssinus received by succinilation and studying of their physical, chemical and immunologic properties.Materials and methods. In the modification species determined the protein content, isoelectric points of protein components by a method of isoelectrofocusing (IEF), esterase activity. Antigenic properties of allergoid studied by methods of an immunodiffusion and an immunoelectrophoresis. Applied microdot immunoenzyme assay to characterize the specific activity of a resulting allergoid. Received by a succinilation allergoid was also studied on physical and chemical indicators.Results. It is established That the formalinization is resulted by the expressed depression of allergenic activity of an allergoid. It is also shown that succinilation leads to essential depression of allergenicity of a monomeric allergoid due to blockade of B-cellular epitopes and to conservation of an adjuvanticity helps to T-cellular epitopes.Conclusion. Thanks to the proved depression of allergenicity and rising of an adjuvanticity monomeric and polymeric allergoid can be recommended for carrying out allergenspecific immunotherapy (ASIT).

PI Determination of Native Proteins In Biological Samples.

The electrophoretic mobility of a protein on an immobilized pH-gradient gel (IPG) depends upon its overall positive (acidic) or negative (basic) charge, the principle underlying the IEF technique. In isoelectrofocusing (IEF), a protein with a net positive or negative charge migrates through the pH gradient gel until it reaches the isoelectric point (pI), a pH at which it remains neutral. Thus, the pI of a protein indicates its net charge, a critical determinant of its stability/activity in a given milieu. Conventionally, the first-dimensional IPG-IEF is followed by a second dimension, by which the focused proteins are denatured/reduced and resolved on an SDS-PAGE gel for subsequent immunoblotting to verify the protein identity. The recent development of one-dimensional, vertical IEF followed by immunoblotting enabled concurrent analysis (pI determination) of multiple samples. The protocol described here outlines vertical IEF and immunoblotting under non-denaturing conditions to determine the pI of native proteins in biological samples. © 2019 by John Wiley & Sons, Inc.

A non-canonical phosphorylation site in β-casein A from non-Mediterranean water buffalo makes quantifiable the adulteration of Italian milk with foreign material by combined isoelectrofocusing-immunoblotting procedures.

The need of controlling illegal addition of water buffalo (WB) milk from foreign countries to the Italian counterpart devoted to the production of Protected Denomination of Origin (PDO) Mozzarella di Bufala Campana (MBC) cheese has promoted the development of simple, fast and cheap isoelectrofocusing (IEF) methods for evaluating the nature of the raw material to be used according to a high-throughput sample multiplexing format, avoiding the use of dedicated mass spectrometry-based procedures. Thus, combined proteomic methods were here integrated with optimized western blotting protocols in solving the complex IEF pattern of casein (CN) mixtures observed when Italian and foreign WB milk are mixed together. Identification of internally deleted αs1-CN hepta-phosphorylated species as well as of still unknown β-CN A hexa-phosphorylated and N-terminally-nicked β-CN A phosphorylated forms present uniquely in foreign WB milk samples, allowed recognizing these molecules as adulteration markers to be assayed in combined IEF-immunoblotting procedures; the latter ones showing optimal migration characteristics to be used in routine assays. A linear relationship between detected area of specific immunorecognized gel bands and percentage of international WB milk added to the Italian counterpart was verified, demonstrating that this method has an adulteration detection limit close to 3% v/v. Based on these results, this analytical procedure is here proposed as optimal one for evaluating the authenticity of PDO MBC cheese products.

Detection of Selenoproteins by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP MS) in Immobilized pH Gradient (IPG) Strips.

In contrast to other trace elements that are cofactors of enzymes and removed from proteins under denaturing conditions, Se is covalently bound to proteins when incorporated into selenoproteins, since it is a component of selenocysteine aminoacid. It implies that selenoproteins can undergo several biochemical separation methods in stringent and chaotropic conditions and still maintain the presence of selenium in the primary sequence. This feature has been used to develop a method for the detection of trace levels of human selenoproteins in cell extracts without the use of radioactive isotopes. The selenoproteins are separated as a function of their isoelectric point (pI) using iso-electrofocusing (IEF) electrophoretic strips and detected by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). This method, therefore referred to as IEF-LA-ICP MS, allowed the detection of several selenoproteins in human cell lines, including Gpx1, Gpx4, TXNRD1, TXNRD2, and SELENOF.

Combined use of Kappa Free Light Chain Index and Isoelectrofocusing of Cerebro-Spinal Fluid in Diagnosing Multiple Sclerosis: Performances and Costs.

Isoelectrofocusing (IEF) to detect oligoclonal bands (OBCs) in cerebrospinal fluid (CSF) is the gold standard approach for evaluating intrathecal immunoglobulin synthesis in multiple sclerosis (MS) but the kappa free light chain index (KFLCi) is emerging as an alternative marker, and the combined/sequential uses of IEF and KFLCi have never been challenged. CSF and serum albumin, IgG, kFLC and lFLC were measured by nephelometry; albumin, IgG and kFLC quotients as well as Link and kFLC indexes were calculated; OCBs were evaluated by immunofixation. A total of 150 consecutive patients: 48 with MS, 32 with other neurological inflammatory diseases (NID), 62 with neurological non-inflammatory diseases (NNID), and 8 without any detectable neurological disease (NND) were investigated. Both IEF and KFLCi showed a similar accuracy as diagnostic tests for multiple sclerosis. The high sensitivity and specificity associated with the lower cost of KFLCi suggested to use this test first, followed by IEF as a confirmative procedure. The sequential use of IEF and KFLCi showed high diagnostic efficiency with cost reduction of 43 and 21%, if compared to the contemporary use of both tests, or the unique use of IEF in all patients. The "sequential testing" using KFLCi followed by IEF in MS represents an optimal procedure with accurate performance and lower costs.

Galactose Epimerase Deficiency: Expanding the Phenotype.

Galactose epimerase deficiency is an inborn error of metabolism due to uridine diphosphate-galactose-4'-epimerase (GALE) deficiency. We report the clinical presentation, genetic and biochemical studies in two siblings with generalized GALE deficiency.Patient 1: The first child was born with a dysmorphic syndrome. Failure to thrive was noticed during the first year. Episodes of heart failure due to dilated cardiomyopathy, followed by liver failure, occurred between 12 and 42 months. The finding of a serum transferrin isoelectrofocusing (IEF) type 1 pattern led to the suspicion of a congenital disorder of glycosylation (CDG). Follow-up disclosed psychomotor disability, deafness, and nuclear cataracts.Patient 2: The sibling of patient 1 was born with short limbs and hip dysplasia. She is deceased in the neonatal period due to intraventricular hemorrhage in the context of liver failure. Investigation disclosed galactosuria and normal transferrin glycosylation.Next-generation sequence panel analysis for CDG syndrome revealed the previously reported c.280G>A (p.[V94M]) homozygous mutation in the GALE gene. Enzymatic studies in erythrocytes (patient 1) and fibroblasts (patients 1 and 2) revealed markedly reduced GALE activity confirming generalized GALE deficiency. This report describes the fourth family with generalized GALE deficiency, expanding the clinical spectrum of this disorder, since major cardiac involvement has not been reported before.

Is it worth to report the presence of a single and additional band in the cerebrospinal fluid detected by isoelectrofocusing?

Despite the revisions of the Mac Donald criteria of multiple sclerosis (MS) in 2010, the cerebrospinal fluid (CSF) analysis by isoelectrofocusing (IEF) remains useful for atypical presentations of MS. The IEF is considered as positive when at least two or more additional bands are detected in the CSF by comparison with the patient's serum but sometimes, the IEF interpretation is more difficult. The goal of our study was to determine the significance when a single band in the CSF is detected by IEF. We conducted a retrospective study on 990 patients who underwent a lumbar puncture followed by a CSF analysis by IEF. Only 2% display such IEF profile (i.e. single and additional band in the CSF). A diagnosis of clinically isolated syndrome or MS was evidenced in 4 among those 21 patients. In conclusion, our data suggest that even if the presence of a single and additional band in the CSF is a rare situation, it should be mentioned to clinicians to not exclude the hypothesis of an inflammatory demyelinating disease of the central nervous system.

The Diagnostic Value of Alpha-1-Antitrypsin Phenotype in Patients with Granulomatosis with Polyangiitis.

The deficiency of alpha-1 protease inhibitor, or alpha-1-antitrypsin (A1AT), predisposes to chronic lung diseases and extrapulmonary pathology. Besides classical manifestations, such as pulmonary emphysema and liver disease, alpha-1-antitrypsin deficiency (A1ATD) is also known to be associated with granulomatosis with polyangiitis (GPA or Wegener's granulomatosis). The aim of our study was to evaluate the frequency of allelic isoforms of A1AT and their clinical significance among GPA patients. Detailed clinical information, including Birmingham Vasculitis Activity Score (BVAS), incidence of lung involvement, anti-proteinase 3 (PR3) antibodies concentrations, and other laboratory data were collected in 38 GPA patients. We also studied serum samples obtained from 46 healthy donors. In all collected samples A1AT phenotyping by isoelectrofocusing (IEF) and turbidimetric A1AT measurement were performed. Abnormal A1AT variants were found in 18.4% (7/38) of cases: 1 ZZ, 4 MZ, 2 MF, and only 1 MZ in control group (2%). The mean A1AT concentration in samples with atypical A1AT phenotypes was significantly lower (P = 0.0038) than in normal A1AT phenotype. We found that patients with abnormal A1AT phenotypes had significantly higher vasculitis activity (BVAS) as well as anti-PR3 antibodies concentration. We conclude that A1AT deficiency should be considered in all patients with GPA.

Phenotypic Expansion of Congenital Disorder of Glycosylation Due to SRD5A3 Null Mutation.

We present a boy, admitted at 4 months, with facial dysmorphism, hypertrichosis, loose skin, bilateral inguinal hernia, severe hypotonia, psychomotor disability, seizures with hypsarrhythmia (West syndrome), hepatosplenomegaly, increased serum transaminases, iris coloboma, glaucoma, corneal clouding and bilateral dilated lateral ventricles, and extra-axial post-cerebellar space. Serum transferrin isoelectrofocusing (IEF) showed a type 1 pattern. Whole-exome genotyping showed a previously reported homozygous nonsense mutation c.320G>A; p.Trp107X in SRD5A3. Epilepsy and glaucoma have been reported only once in the 19 described SRD5A3-congenital glycosylation defect patients, and corneal clouding not at all.

Abstract 833: Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol

Abstract There are seven β-tubulin isotypes present in distinct quantities in cells of different origin. Altered expression of β-tubulin isotypes were found in cells resistant to microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, ixabepilone and discodermolide, all of which bind to β-tubulin. For example, the level of βIII-tubulin was very low in the human ovarian cancer cell line, Hey, but increased 8- to 15-fold in two drug resistant daughter cell lines, EpoB8 and Ixab80, respectively. The βV-tubulin level was increased by 2-fold in EpoB8 cells. In contrast, levels of βI- and βIV-tubulin were unchanged and decreased, respectively, in EpoB8. Therefore, it is important to measure the binding affinity of MSAs to different β-tubulin isotypes and study their influence on the drug resistant phenotype. To study the relative binding affinities of MSAs, tubulins from HeLa cells, bovine brain, porcine brain and chicken erythrocytes were specifically photolabeled with the tritium labeled 2-(m-azidobenzoyl)taxol, in the presence and absence of Taxol, Taxotere, epothilone B, ixabepilone and discodermolide. β-tubulin isotype content from these sources was different, particularly for chicken erythrocytes that contain only one β-tubulin isotype, βVI. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulins from different sources. Taxol had a minimal inhibitory effect on HeLa, bovine and porcine brain β-tubulin, but a strong inhibitory effect on chicken erythrocyte β-tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. However, Taxotere appeared to have a stronger inhibitory effect than Taxol on photolabeling of β-tubulins from all sources tested. Ixabepilone had the strongest inhibitory effect on HeLa β-tubulin. The inhibition pattern for β-tubulin from porcine brain correlated very well with that from bovine brain. To determine drug binding affinities for the different β-tubulin isotypes, tubulins from different sources were photolabeled and the isotypes resolved by high resolution isoelectrofocusing (IEF). Approximately 20 bands were seen with bovine and porcine brain tubulin, but only 3-5 bands were resolved with chicken erythrocyte tubulin. Many bands represent posttranslationally modified tubulins. IEF patterns for bovine and porcine brain tubulin were very similar. All bands were analyzed by mass spectrometry following CNBr digestion, and the identity and relative quantity of each β-tubulin isotype determined. After measuring the radioactivity associated with each band, it was found that the binding affinity of 2-(m-azidobenzoyl)taxol to βIII-tubulin was the lowest, compared to other β-tubulin isotypes. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2014-833

Assessment of the reproducibility of oligoclonal IgM band detection for its application in daily clinical practice

BackgroundThe presence of oligoclonal IgM bands (OCMB) in cerebrospinal fluid (CSF) is an unfavourable prognostic marker in multiple sclerosis. There is no commercial test to investigate OCMB status. However, a sensitive and specific isoelectrofocusing (IEF) and western blot method was described. We aimed to study the inter-centre reproducibility of this technique, a necessary condition for a reliable test to be incorporated into clinical practice. MethodsThe presence of OCMB was analysed by IEF and western blot with prior reduction of pentameric IgM. We assayed the reproducibility of this test in a blinded multicentre study performed in 13 university hospitals. Paired-CSF and serum samples from 52 neurological patients were assayed at every centre. ResultsGlobal analysis rendered a concordance of 89.8% with a kappa value of 0.71. ConclusionThese data indicate that OCMB detection by means of IEF and western blot with IgM reduction shows a good interlaboratory reproducibility and thus can be used in daily clinical setting.

Speciation analysis for trace levels of selenoproteins in cultured human cells.

Our paper presents the development of a semi-quantitative method for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts; it offers a first comprehensive screening of the entire biological selenoproteomes expressed in cell lines without the use of radioactive (75)Se. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL(-1) (as Se); by far the lowest ever reported. The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). The IEF-LA-ICPMS indicates the presence of multiple forms of some selenoproteins which are for the moment impossible to distinguish because of the similarity of the bottom-up, proteomics data sets.

Large-scale speciation of selenium in rice proteins using ICP-MS assisted electrospray MS/MS proteomics

A Se-targeted bottom-up proteomics approach was developed for the identification of Se-containing proteins in rice grown naturally on seleniferous soils. The proteins were separated by 2D gel electrophoresis. The position of Se-containing spots was tentatively identified by the correlation between the 1D isoelectrofocusing (IEF) and 1D SDS electropherograms of a sample aliquot and confirmed by (78)Se imaging in the 2D gel. The method was complemented by the ICP-MS assisted shotgun proteomics approach. The proteins were identified by capHPLC with the dual ICP MS and electrospray Orbitrap MS detection. The first ever comprehensive study of rice selenoproteome revealed the presence of selenium, as both selenomethionine (SeMet) and selenocysteine (SeCys) residues, in a dozen proteins including a 19 kDa globulin, granule-bound starch synthase, and the family of glutelin-type seed storage proteins.

Hb Grove City [β38(C4)Thr→Ser, ACC>AGC; HBB: c.116C>G]: A New Low Oxygen Affinity β Chain Variant

A previously unreported β chain hemoglobin (Hb) variant, Hb Grove City [β38(C4)Thr→Ser, ACC>AGC; HBB: c.116C>G], was discovered in a woman who presented with hypoxia and mild anemia. Her young daughter also tested positive for the variant and displayed similar symptoms. Hemoglobin-oxygen dissociation testing confirmed right-shifted oxygen dissociation curves. A corresponding Hb variant was detected by high performance liquid chromatography (HPLC) and intact mass spectrometry (MS) but was not detected by capillary electrophoresis (CE), isoelectrofocusing (IEF) or alkaline or acid electrophoresis. DNA sequencing analysis confirmed a β-globin gene mutation. All three previous mutations at this locus affect oxygen affinity, as does this new variant. This newly described variant showed variable stability results and therefore may be mildly unstable but is not associated with microcytosis, significant hemolysis or clinically evident cyanosis. It is important to consider hemoglobinopathies in patients who are anemic and have unexplained hypoxia. Arterial blood gas and p50 evaluations may prevent unnecessary diagnostic interventions. Additionally, Hb variants with altered oxygen affinity can be electrophoretically silent; therefore, multiple methods including MS and/or DNA sequencing are warranted when clinical suspicion is high.

English

Medicago truncatula leaves were used as the experimental materials. Total proteins of leaves were extracted by trichloracetic acid (TCA)-acetone method and proteins had a better separation using gel strips, forming an immobilized non-linear 3 to 10 pH gradient focusing 123,000 vhr combined with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie Brilliant Blue G-250 and digitalized gels were analyzed using the PDquest 8.0.1 software. The results indicated that 931 protein dots were detected in the gel. A technology suitable for the M. truncatula leaves protein extraction by TCA/acetone and a protocol for two-dimensional electrophoresis (2-DE) was established, which provides technical support for M. truncatula leaf proteome research. Key words: Medicago truncatula, proteome, two-dimensional polyacrylamide gel electrophoresis (2-DE), isoelectrofocusing (IEF).

Bovine &amp;lt;i&amp;gt;β&amp;lt;/i&amp;gt;-casein: Detection of two single nucleotide polymorphisms by bidirectional allele specific polymerase chain reaction (BAS-PCR) and monitoring of their variation

Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platforms are available for semiautomated or fully automated SNP geno-typing. However, in some cases the use of simple and rather cheap methods is an effective tool to be implemented within one’s own laboratory for the routine analysis of a specific SNP. The present paper describes two simple tests based on the bidirectional allele-specific polymerase chain reaction (BAS-PCR) developed for the identification of β-casein (CSN2) B and I genetic variants. The practical application of the two methods on a panel of 84 Italian Brown bulls and 100 Italian Friesian cows is also discussed, including the biological significance of the two genetic variants and the importance of taking their occurrence into account when linkage analyses are performed on milk functional properties. A combined system for analysing milk protein variants by isoelectrofocusing (IEF) and the BAS-PCR assay developed for CSN2*I is described.

Forensic aspects of postmortem serum carbohydrate-deficient transferrin analysis as a marker of alcohol abuse

Carbohydrate-deficient transferrin (CDT) has been suggested as one of alcohol abuse indicators having produced good results in forensic medicine for years. The aim of the study was to identify correlation between present methodology of alcohol abuse diagnosis at autopsy (macroscopic and microscopic findings) and CDT examination using the method of isoelectrofocusing (IEF) in polyacrylamide gel electrophoresis (PAGE). We also analyzed if the time interval between the moment of death and blood sample collection influences CDT findings. The method used for CDTanalysis was IEF-PAGE. Sera of 49 males and 11 females aged 14-87 years, average age 46.85+/-18.53, were used in this study. Control group consisted of five patients who died after medical treatment that lasted longer than 15 days, and five patients who started Disulfiram therapy in controlled hospital environment. The results obtained in CDT examination in dead bodies' sera showed sensitivity 59% and specificity 71%. A high incidence of falsely positive CDT result was noticed in liver failure and cirrhosis of non-alcoholic origin. CDT analysis is also possible to be done in samples collected postmortem up to 76 hours. In forensic medicine, the method of CDT determination is reliable for the diagnosis of alcohol abuse.

Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4 pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1 ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4 ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.