Secreted phosphoglucose isomerase is a novel biomarker of nonalcoholic fatty liver in mice and humans.

The current gold standard for diagnosis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is through a liver biopsy, and there is an urgent need to develop non-invasive methods for early detection. We previously demonstrated metabolic remodeling in the mouse fatty liver, which is marked by increased hepatic expression and activities of phosphoglucose isomerase (PGI) and several other glycolytic enzymes. Since PGI is actively transported out of the cell, acting as a multifunctional cytokine referred to as autocrine motility factor (AMF), we explored the possibility that PGI secreted from the fatty liver may be targeted for early detection of the silent disease. We report here that mice with NASH exhibited significantly elevated serum PGI enzyme activities compared to normal control (P < 0.005). We further confirmed the finding using serum/plasma samples (n = 73) collected from a cohort of NASH patients who were diagnosed according to Kleiner's criteria, showing a normal mean PGI of 19.5 ± 8.8 IU/L and patient mean PGI of 105.6 ± 79.9 IU/L (P < 0.005). In addition, elevated blood PGI in NASH patients coincided with increased blood L-lactate. Cell culture experiments were then conducted to delineate the PGI-lactate axis, which revealed that treatment of HepG2 cells with recombinant PGI protein stimulated glycolysis and lactate output, suggesting that the disease-induced PGI likely contributed to the increased lactate in NASH patients. Taken together, the preclinical and clinical data validate secreted PGI as a useful biomarker of the fatty liver that can be easily screened at the point of care.

Glucose-6-phosphate isomerase is associated with disease activity and declines in response to infliximab treatment in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA), a systemic autoimmune disease characterized by synovial inflammation, can cause cartilage and bone damage as well as disability. The aim of this study was to explore whether serum glucose-6-phosphate isomerase (GPI) is correlated with disease activity and the value of GPI in the evaluation of infliximab treatment in patients with RA. METHODS: Sixty-two patients with RA who had an inadequate response to methotrexate (MTX) were enrolled in Peking University People's Hospital from July 1, 2016 to July 31, 2018. Infliximab (3 mg/kg, intravenous at weeks 0, 2, and 6 and then every 8 weeks) was administered to patients with stable background MTX therapy. Serum samples were obtained at baseline and week 18. Serum GPI levels were determined using enzyme-linked immunosorbent assay. The associations between serum GPI levels and clinical features were analyzed. RESULTS: Serum GPI was positively correlated with Disease Activity Score in 28 joints (DAS28), swollen joint count, tender joint count and C-reactive protein level (P < 0.001, P < 0.001, P < 0.001, and P = 0.033, respectively). The change of DAS28 in GPI-positive patients was greater than that in GPI-negative patients (P < 0.001). Compared with those for patients receiving MTX monotherapy at baseline, the GPI levels were significantly declined when MTX was combined with infliximab (P < 0.001). CONCLUSION: Serum GPI is related to disease activity and clinical response to infliximab treatment.

A Retrospective Study: The Significance of Combined Testing of Serum Markers for Diagnosis of Rheumatoid Arthritis.

BACKGROUND There have been few studies on the value of various antibody combinations in rheumatoid arthritis (RA) diagnosis, and a lack of studies with large sample sizes, especially in the Chinese population. This study retrospectively evaluated the diagnostic value of a combined assay of five auto-antibodies [anti-cyclic citrullinated peptide (anti-CCP), anti-keratin (AKA), anti-RA 33, glucose-6-phosphate isomerase (GPI), and rheumatoid factor (RF)] for RA. MATERIAL AND METHODS Data were obtained from 5,725 patients with rheumatic diseases in Southwest Hospital of Chongqing from 2011 to 2014. Detection of the five serological markers was performed for all study patients using the appropriate method for each antibody. RESULTS It was found that of the 5,725 patients, the positive rates for RF, anti-CCP, anti-RA 33, AKA, and GPI were 52.5%, 40.1%, 12.8%, 12.0%, and 50.0% respectively. In RA patients, the positive rates were 83.3%, 68.5%, 16.6%, 20.8%, and 77.9% respectively, which were all significantly higher than those detected in patients with the other diseases (p<0.01). The areas under the receiver operator characteristic (ROC) curve for RF, anti-CCP, anti-RA 33, AKA, and GPI were 0.857, 0.831, 0.528, 0.602, and 0.822 respectively, indicating that these five serological markers display favorable diagnostic value for RA. There were positive correlations between anti-CCP antibody and RF and GPI (p<0.01) and between RF and GPI (p<0.01), but no correlation between anti-RA 33 and AKA (p<0.01). The specificity of the combination of anti-CCP, AKA, and GPI was 100% for RA diagnosis. CONCLUSIONS The combined assay of serological markers significantly improved the diagnostic specificity for RA. The diagnostic value of RF for RA was the highest and the combined assay for anti-CCP, AKA, and GPI had the highest specificity for RA diagnosis.

Carbohydrate-based electrochemical biosensor for detection of a cancer biomarker in human plasma.

Autocrine motility factor (AMF) is a tumor-secreted cytokine that stimulates tumor cell motility in vitro and metastasis in vivo. AMF could be detected in serum or urine of cancer patients with worse prognosis. Reported as a cancer biomarker, AMF secretion into body fluids might be closely related to metastases formation. In this study, a sensitive and specific carbohydrate-based electrochemical biosensor was designed for the detection and quantification of a protein model of AMF, namely phosphoglucose isomerase from rabbit muscle (RmPGI). Indeed, RmPGI displays high homology with AMF and has been shown to have AMF activity. The biosensor was constructed by covalent binding of the enzyme substrate d-fructose 6-phosphate (F6P). Immobilization was achieved on a gold surface electrode following a bottom-up approach through an aminated surface obtained by electrochemical patterning of ethylene diamine and terminal amine polyethylene glycol chain to prevent non-specific interactions. Carbohydrate-protein interactions were quantified in a range of 10 fM to 100nM. Complex formation was analyzed through monitoring of the redox couple Fe2+/Fe3+ by electrochemical impedance spectroscopy and square wave voltammetry. The F6P-biosensor demonstrates a detection limit of 6.6 fM and high selectivity when compared to other non-specific glycolytic proteins such as d-glucose-6-phosphate dehydrogenase. Detection of protein in spiked plasma was demonstrated and accuracy of 95% is obtained compared to result obtained in PBS (phosphate buffered saline). F6P-biosensor is a very promising proof of concept required for the design of a carbohydrate-based electrochemical biosensor using the enzyme substrate as bioreceptor. Such biosensor could be generalized to detect other protein biomarkers of interest.

[Significance of antibodies to the citrullinated glucose-6-phosphate isomerase peptides in rheumatoid arthritis].

OBJECTIVE: To detect the anti-citrullinated glucose-6-phosphate isomerase (GPI) 70-88 peptide antibody (anti-C-GPI(70-88) antibody), anti-citrullinated GPI 435-453 peptide antibody (anti-C-GPI(435-453) antibody), anti-GPI 70-88 peptide antibody (anti-GPI(70-88) antibody) and anti-GPI 435-453 peptide antibody(anti-GPI(435-453) antibody) in the serum of rheumatoid arthritis (RA) patients, and examine the diagnostic values of the anti-C-GPI peptide antibodies in RA. METHODS: The anti-C-GPI(70-88) antibody, anti-C-GPI(435-453) antibody, anti-GPI(70-88) antibody and anti-GPI(435-453) antibody were detected by enzyme-linked immunosorbent assay (ELISA) in 191 RA patients, 129 other rheumatic diseases and 74 healthy controls. The clinical and laboratory data of the patients with RA were collected, and the values of anti-C-GPI peptide antibodies in the diagnosis of RA and the relationships of anti-C-GPI peptide antibodies with the clinical and laboratory parameters analyzed. RESULTS: (1) The mean titers of the anti-C-GPI(70-88) antibody and the anti-C-GPI(435-453) antibody in the RA patients (respectively, 68.71 ± 4.20 and 51.78 ± 3.13) were significantly higher than those with other rheumatic diseases and healthy individuals (P <0.05). However, the mean titers of the anti-GPI(70-88) antibody and anti-GPI(435-453) antibody in the RA patients were similar to those with other rheumatic diseases and healthy individuals. (2) The diagnostic sensitivity and specificity of the anti-C-GPI(70-88) antibody for RA were 41.88% and 84.50% respectively; and the diagnostic sensitivity and specificity of the anti-C-GPI(435-453) antibody for RA were 46.05% and 86.05% respectively. The sensitivity of combined detection of the two anti-C-GPI peptide antibodies was 50.79%, and the specificity was 81.40%. (3) The positive rates of the anti-C-GPI(70-88) antibody and the anti-C-GPI(435-453) antibody were 35% and 45% respectively in those patients with negative anti-cyclic citrullinated peptide antibody, anti-keratin antibody, and rheumatoid factor. (4) There was no significant difference in clinical and laboratory indicators between the anti-C-GPI(70-88) antibody or anti-C-GPI(435-453) antibody positive group and negative group. CONCLUSION: The anti-C-GPI(70-88) antibody and anti-C-GPI(435-453) antibody can be detected in the serum of RA patients, and C-GPI may be involved in the pathogenesis of RA. There is a certain diagnostic significance for the sera-negative RA.

[Significance of glucose-6-phosphate isomerase assay in early diagnosis of rheumatoid arthritis].

OBJECTIVE: To explore the titer of glucose-6-phosphate isomerase (GPI) for early diagnosis of the outpatient with rheumatoid arthritis (RA) in real life, and to analyze its relationship with disease activity. METHODS: In the study, 1 051 patients with arthritis were collected in the group who had joints tender and swelling, and 90 cases of healthy people as a control group. ELISA method was used to detect the serum level of GPI, and according to clinical features and laboratory test, all the patients including 525 RA patients, the other patients including osteoarthritis (OA), 134 cases of seronegative spine joint disease (SpA), 104 cases of systemic lupus erythematosus (SLE), 31 cases of primary Sjogren syndrome (pSS), 24 cases of gout arthritis (GA), 22 cases of other connective tissue diseases (including polymyalgia rheumatica, dermatomyositis, systemic sclerosis, adult Still disease) and 46 cases of other diseases (including 165 cases of osteoporosis, avascular necrosis of the femoral head, traumatic osteomyelitis, bone and joint disease, juvenile rheumatoid arthritis, tumor). The diagnostic values of GPI were assessed, and the differences between the GPI positive and negative groups of the RA patients in clinical characteristics, disease activity, severity and inflammatory index analyzed. RESULTS: The positive rate of serum GPI in the patients with RA was 55.4%, contrasting to other autoimmune diseases (14.3%) and healthy controls (7.78%)(P<0.001). Compared with the OA and SpA patients, the RA group was increased more significantly, and the difference was statistically significant (P<0.001). The diagnostic value of GPI alone for RA was 0.39 mg/L, the sensitivity was 54.2%, and specificity was 87.3%. The positive rate of GPI in RF negative patients was 36.1%; the positive rate of GPI in anti-CCP antibody negative patients was 34.2%; the positive rate of GPI in RF and anti-CCP antibody negative patients was 24.1%. The level of GPI had positive correlation (P<0.05) with ESR, RF, anti-CCP antibody and HRF-IgG. CONCLUSION: GPI is sensitive in the patients with RA; GPI positive is important in the diagnosis of RA with anti-CCP antibody and/or RF negative patients. The titer of GPI is related with disease activity of RA.

New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10(-6)). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests.

Macrophage scavenger receptor 1 (Msr1, SR-A) influences B cell autoimmunity by regulating soluble autoantigen concentration.

The class A macrophage scavenger receptor Msr1 (SR-A, CD204) has been reported to participate in the maintenance of immunological tolerance. We investigated the role of Msr1 in a mouse model of autoantibody-dependent arthritis. Genetic deficiency of Msr1 in K/BxN TCR transgenic mice decreased the incidence and severity of arthritis because of decreased autoantibody production. Despite normal initial activation of autoreactive CD4(+) T cells, potentially autoreactive B cells in Msr1(-/-) K/BxN mice retained a naive phenotype and did not expand. This was not due to an intrinsic B cell defect. Rather, we found that macrophages lacking Msr1 were inefficient at taking up the key autoantigen glucose-6-phosphate isomerase and that Msr1-deficient mice had elevated serum concentrations of glucose-6-phosphate isomerase. Arthritis developed normally when bone marrow from Msr1(-/-) K/BxN mice was transplanted into hosts whose macrophages did express Msr1. Thus, Msr1 can regulate the concentration of a soluble autoantigen. In this model, the absence of Msr1 led to higher levels of soluble autoantigen and protected mice from developing pathogenic autoantibodies, likely because of altered cognate interactions of autoreactive T and B cells with impaired differentiation of follicular Th cells.

[Value of four serum markers in the diagnosis of rheumatoid arthritis].

OBJECTIVE: To systematically evaluate the values of 4 serum markers in the diagnosis of rheumatoid arthritis (RA). METHODS: Serum samples were obtained from 278 RA patients and 510 control subjects and the levels of rheumatoid factor (RF), anticyclic citrullinated peptide antibody (CCP), antikeratin antibody (AKA), and glucose-6-phosphate isomerase (GPI) were detected using immune turbidimetry, ELISA, indirect immunofluorescence, and ELISA, respectively. The values of these 4 serum markers and their combinations in RA diagnosis were systemically assessed. RESULTS: In RA diagnosis using one serum marker, two markers, and three or four markers, RF, RF+CCP, RF+CCP+GPI, respectively, had the highest sensitivity; CCP, CCP+AKA, and RF+CCP+AKA+GPI, respectively, had the highest specificity; CCP, CCP+GPI, and RF+CCP+AKA+GPI, respectively, had the highest positive predictive value; GPI, RF+CCP, and RF+CCP+GPI, respectively, had the highest negative predictive value; CCP, CCP+GPI, and RF+CCP+AKA+GPI, respectively, had the highest positive likely ratio; GPI, RF+CCP, and RF+CCP+GPI, respectively, had the lowest negative likely ratio. CONCLUSION: CCP, RF+CCP, and RF+CCP+GPI are the most ideal for RA diagnosis using one, two, and three or more markers, respectively. CCP is the essential marker for RA diagnosis, and a combined detection of the serum makers can significantly improve the diagnostic accuracy.

Anti-citrullinated glucose-6-phosphate isomerase peptide antibodies in patients with rheumatoid arthritis are associated with HLA-DRB1 shared epitope alleles and disease activity.

To identify and characterize anti-citrullinated glucose-6-phosphate isomerase (GPI) peptide antibodies in patients with rheumatoid arthritis (RA). Nine GPI arginine-bearing peptides in human GPI protein were selected and cyclic citrullinated GPI peptides (CCG-1-9) were constructed. Samples were obtained from RA (n = 208), systemic lupus erythematosus (SLE) (n = 101), Sjögren's syndrome (SS; n = 101) and healthy controls (n = 174). Antibodies against CCG-1-9 were measured, and anti-citrullinated α-enolase-1 (CEP-1), -cyclic citrullinated peptides (CCP) and -GPI proteins antibodies were also examined. Patients with RA were genotyped for HLA-DRB1. The numbers of shared epitope (SE) alleles were counted and compared with those of the autoantibodies. Rabbit GPI was citrullinated with rabbit peptidylarginine deiminase and immunoblot analysis of RA sera performed. The levels of autoantibodies were compared before and after treatment with TNF antagonists in 58 RA patients. Anti-CCG-2, -4 and -7 antibodies were detected in 25·5, 33·2 and 37·0% patients with RA, respectively, and these antibodies were very specific for RA (specificity, 98·1-99·7%). Altogether, 44·2, 86·1 and 13·9% of RA sera were positive for anti-CEP-1, -CCP and -GPI protein antibodies, respectively. Anti-CCG-2, -4 and -7 antibodies were correlated with anti-CCP and anti-CEP-1 antibodies and with the presence of HLA-DRB1 SE alleles. Citrullinated GPI protein was detected using RA sera. Treatment with tumour necrosis factor antagonists reduced significantly the levels of anti-CCG-2 and -7 but not of anti-CEP-1 antibodies. This is the first report documenting the presence of anti-CCG antibodies in RA. Anti-CCG-2 and -7 antibodies could be considered as markers for the diagnosis of RA and its disease activity.

Genetic structure and variation of Van cats.

To determine the genetic structure and variation of Van cats and some other cats, seven enzyme loci were examined using horizontal starch gel electrophoresis. ME bands were observed for the first time in cats. For the enzyme loci CA ( 1 ), SOD, GPI, and GOT, neither the individual Van cats nor the specimens of other cat species exhibited any variation. These enzymes presented identical bands, all of which were homozygous. With respect to the PGD, ME, and ESD loci, however, genetic variation was observed in all of the cats. Hence, three of the seven gene-enzyme systems (43%) were polymorphic with two alleles, contributing to an estimate of average heterozygosity of 0.33-0.49 for the Van cats. PGD was the most discriminatory among the three polymorphic loci. The phylogenetic tree indicated that the Van, Persian, Turkish Angora, and Turkish Tekir cats are distinct from Siamese and Bombay cats.

Diagnostic value of glucose-6-phosphate isomerase in rheumatoid arthritis.

BACKGROUND: Although glucose-6-phosphate isomerase (G6PI), anti-G6PI antibodies and G6PI-containing immune complexes (G6PI-CIC) have proved high expression in patients with rheumatoid arthritis (RA), comprehensive evaluation of the G6PI-derived markers, G6PI antigen, anti-G6PI Abs, G6PI-CIC and G6PI mRNA, in the diagnosis of RA remains necessary. METHODS: We measured G6PI antigen, anti-G6PI Abs, C1q/G6PI-CIC as well as anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) in serum and concomitantly synovial fluid (SF) by ELISA in RA, other rheumatic diseases and healthy controls. The G6PI mRNA expression in peripheral blood mononuclear cells (PBMCs) was assessed with real-time PCR. RESULTS: As compared with non-RA patients, RA patients had increased levels of G6PI antigen, anti-G6PI Abs, C1q/G6PI-CIC and G6PI mRNA expression in sera or PBMCs, and increased levels of G6PI and C1q/G6PI-CIC in SF. The serum G6PI levels in RA patients positively correlated with anti-G6PI Abs, C1q/G6PI-CIC, G6PI mRNA, anti-CCP Abs, RF, CRP and ESR, respectively. The area under curve analyses demonstrated that serum G6PI had the best discriminating power for RA and active RA followed by C1q/G6PI-CIC, anti-G6PI Abs and G6PI mRNA. The simultaneous use of serum G6PI and anti-CCP Abs assays in the form of either of them tested positive gave improved sensitivities of 88.1% for RA and 95.8% for active RA. CONCLUSIONS: Despite the elevated expression of all G6PI-derived markers in RA, the serum G6PI has the best discriminating power among the four G6PI-derived markers. The serum G6PI determination either alone or in combination with anti-CCP Abs improves the diagnosis of RA.

Elevated serum glucose-6-phosphate isomerase correlates with histological disease activity and clinical improvement after initiation of therapy in patients with rheumatoid arthritis.

OBJECTIVE: To determine serum glucose-6-phosphate isomerase (GPI) concentrations in patients with rheumatoid arthritis (RA), and to test whether they correlate with objective measures of disease activity. METHODS: Sera from 116 patients with RA, 69 patients with non-RA rheumatic diseases, and 101 healthy controls were analyzed. Levels of soluble serum GPI were measured by ELISA. Histological disease activity was determined with the synovitis score in synovial needle biopsies from 58 of the 116 patients with RA. Thirty-one of the 58 synovium samples were stained for CD68, CD3, CD20, CD38, CD79a, and CD34 by immunohistochemistry. Demographic data were collected, as well as serological and clinical variables that indicate RA disease activity, for Spearman correlation analysis. RESULTS: Serum GPI level correlated positively with the synovitis score (r = 0.278, p = 0.034). Significantly higher soluble GPI levels were detected in the RA sera compared with sera from healthy controls and the non-RA disease controls (2.25 ± 2.82 vs 0.03 ± 0.05 and 0.19 ± 0.57 µg/ml, respectively; p < 0.0001). The rate of serum GPI positivity was significantly higher in the RA patients than in the non-RA disease controls (64.7% vs 10.1%; p < 0.0001). Spearman analysis showed no significant correlation between serum GPI level and Disease Activity Score in 28 joints at baseline. After initiation of antirheumatic treatments, GPI levels decreased significantly (2.81 ± 3.12 vs 1.44 ± 2.09 µg/ml; p = 0.016), paralleling improvement of the disease activity indices. CONCLUSION: Elevated serum GPI may be involved in the synovitis of RA and may prove useful as a serum marker for disease activity of RA.

[Diagnostic value of glucose-6-phosphate isomerase in rheumatoid arthritis patients: systematic review].

In order to evaluate the diagnostic accuracy of glucose-6-phosphate isomerase in patients with rheumatoid arthritis, retrieval was performed using the data bases of Medline, Embase, Cochrane library, Cmcc and Cbmdisc (1990 to 2007). We included the articles which reported the studies of GPI measured by enzyme-linked immunosorbent assay in the diagnosis of RA patients. Then we reviewed 15 article and used RevMan Software for analysis; the heterogeneity among the articles was determined to be high (chi2 = 191.65, P < 0.00001). When we analyzed the 5 articles wherein serum was used as the standard, we noticed homogeneity (chi2 = 6.97, P = 0.14). The summary sensitivity was 25%; the summary specificity was 80%; the area under the curve was 0.6279. Our study demonstrated that GPI exhibited high specificity and low sensitivity in diagnosing RA cases. We suggest that GPI be used in conjunction with some assay or other that is characterized by high sensitivity.

Development of a comprehensive red blood cell enzymopathy laboratory in Thailand: the study of normal activity in eight erythroenzymes in Thais.

In order to provide a reference range for normal red blood cell enzyme activities in Thai, we analyzed data from 113 healthy non-anemic Thai people (55 males and 58 females) age 1-42 years, who all had a normal pattern of hemoglobin typing (HbA and HbA2 less than 3.5%). Hematological analysis was performed using an automated cell counter and the hemoglobin studies were carried out by low pressure liquid chromatography. Owing to a high frequency of alpha-thalassemia in Thailand, cases with an MCV < 75 fl were excluded from the study since these cases were likely to be heterozygotes for alpha0-thalassemia. Cases with reticulocytes > 2.5% were excluded from the study since reticulocytes have a higher enzyme activity than mature erythrocytes. Cases with abnormal red blood cell morphology, such as spherocytes and ovalocytes, were also excluded. These criteria were applied to select "normal" controls for our analysis. We assayed eight red blood cell enzyme activities in normal subjects: glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), pyruvate kinase (PK), hexokinase (HK), glucose phosphate isomerase (GPI), phosphofructokinase (PFK), aldolase (ALD) and phosphoglycerate kinase (PGK). The mean normal ranges (+/- SD) for G6PD, 6PGD, PK, HK, GPI, PFK, ALD and PGK were 12.7 (+/-2.2), 10.7 (+/-1.3), 18.5 (+/-4.0), 1.5 (+/-0.4), 80.5 (+/-11.8), 11.8 (+/-2.1), 4.5 (+/-1.6) and 370 (+/-43) IU/gHb, respectively. Age-dependent differences for the reference values for these enzyme activities were summarized. All red blood cell enzyme activities were highest during the early childhood period and slightly lower in the adult period. These values will be of clinically useful for future reference.

"Proteotyping": population proteomics of human leukocytes using top down mass spectrometry.

Characterizing combinations of coding polymorphisms (cSNPs), alternative splicing and post-translational modifications (PTMs) on a single protein by standard peptide-based proteomics is challenging owing to <100% sequence coverage and the uncoupling effect of proteolysis on such variations >10-20 residues apart. Because top down MS measures the whole protein, combinations of all the variations affecting primary sequence can be detected as they occur in combination. The protein form generated by all types of variation is here termed the "proteotype", akin to a haplotype at the DNA level. Analysis of proteins from human primary leukocytes harvested from leukoreduction filters using a dual on-line/off-line top down MS strategy produced >600 unique intact masses, 133 of which were identified from 67 unique genes. Utilizing a two-dimensional platform, termed multidimensional protein characterization by automated top down (MudCAT), 108 of the above protein forms were subsequently identified in the absence of MS/MS in 4 days. Additionally, MudCAT enables the quantitation of allele ratios for heterozygotes and PTM occupancies for phosphorylated species. The diversity of the human proteome is embodied in the fact that 32 of the identified proteins harbored cSNPs, PTMs, or were detected as proteolysis products. Among the information were three partially phosphorylated proteins and three proteins heterozygous at known cSNP loci, with evidence for non-1:1 expression ratios obtained for different alleles.

Patients with inflammatory arthritic diseases harbor elevated serum and synovial fluid levels of free and immune-complexed glucose-6-phosphate isomerase (G6PI).

In K/BxN mice, anti-glucose-6-phosphate isomerase (G6PI) IgG antibodies (Abs) cause joint-specific inflammation and destruction. Anti-G6PI Abs are also present in humans with inflammatory arthritis, especially among patients with rheumatoid arthritis (RA). A contributing factor to the induction of such autoantibodies may be upregulated expression of the corresponding antigen G6PI in affected tissues and/or increased levels of G6PI in the circulation. To determine G6PI levels and the presence of free G6PI and/or G6PI-containing immune complexes in sera and synovial fluids (SF) of patients with different arthritides, serum and SF obtained concomitantly from 91 clinically well-defined arthritis patients were assessed in a blinded manner for G6PI enzymatic assay and for G6PI protein concentration by ELISA. Sera and SF from patients with immune-based inflammatory arthritis contained significantly higher levels of G6PI enzymatic activity compared to sera or SF from patients with non-immune-based inflammatory arthritis or healthy controls. In addition, significantly higher levels of total G6PI protein concentration (including both enzymatically active and inactive forms) were present in sera of RA patients vs. those with other immune-based or non-immune-based inflammatory arthritis.G6PI in sera and SF were present both as G6PI-containing immune complexes and as free G6PI, with the majority of free G6PI existing as tetramers with lesser amounts of dimers and monomers. Levels of G6PI enzymatic activity in the sera of most immune-based inflammatory arthritis patients are elevated and may reflect ongoing inflammation and cell destruction. The high serum levels of enzymatically inactive forms of G6PI in RA relative to those in other arthritic diseases are partially due to G6PI-containing immune complexes, a portion of which also contains C1q. Overall, our study supports the notion that elevated G6PI levels present in patients with immune-based inflammatory arthritis may contribute to elevated levels of anti-G6PI Abs and G6PI/anti-G6PI immune complexes. This, in turn, may trigger production of proinflammatory cytokines and perpetuate the inflammatory process.

Potential endocrine function of the glycolytic enzyme glucose-6-phosphate isomerase during implantation.

Glucose-6-phosphate isomerase (GPI) is a glycolytic enzyme that also acts as autocrine motility factor, a secreted protein that stimulates tumor cell motility. We have shown that GPI is required for embryo implantation in the domestic ferret. Here, we tested the hypothesis that GPI is produced and secreted into the bloodstream by ferret luteal cells. Plasma GPI activity increased significantly during the pre-implantation period in both pregnant and pseudopregnant ferrets. Explants from Corpus luteum (CL) and follicles of the pre-implantation period were cultured to ascertain their ability to secrete GPI. The medium of cultured luteal extracts contained significantly more GPI activity than the medium of cultured follicles, a control tissue. GPI activity in the medium increased significantly with increasing pregnancy stage, from pregnancy days 3 to 12. However, GPI activity within explant homogenates was the same in CL and follicles and at all days of pregnancy. Thus, CL but not follicles, secrete GPI during the pre-implantation period. Our findings suggest that GPI may be acting in an endocrine manner, being secreted from the CL into the blood, and acting to promote implantation, which occurs at a distant site, the uterus.

Glucose-6-phosphate isomerase deficiency.

Most of the metabolic needs of erythrocytes are covered by glycolysis, the oxidative pentose phosphate pathway and the glutathione cycle. Hereditary enzyme deficiencies of all these pathways have been identified, among which glucose-6-phosphate isomerase (GPI) deficiency is the second most frequent erythroenzymopathy in glycolysis, being associated with non-spherocytic haemolytic anaemia of variable severity. This autosomal recessive genetic disorder may be associated in some cases with neurological impairment. GPI is a dimeric enzyme that catalyses the reversible interconversion of fructose-6-phosphate and glucose-6-phosphate. Virtually all the mutant gene products reported are characterized by marked instability and normal substrate affinities, but altered catalytic activity and electrophoretic migration rates. At the nucleotide level, 29 mutations have been reported. This chapter reviews (a) the clinical pattern of the condition; (b) biochemical and molecular studies; (c) structure-function relationships; (d) the molecular basis of neurological dysfunctions sometimes associated with GPI deficiency; and (e) the correlation between the severity of the anaemia and the molecular defect.