Volume 40, Issue 1

Myeloperoxidase (MPO) is a glycoprotein released by activated polymorphonuclear neutrophils, which takes part in the defense of the organism through production of hypochlorous acid (HOCl), a potent oxidant. Since the discovery of MPO deficiency, initially regarded as rare and restricted to patients suffering from severe infections, MPO has attracted clinical attention. The development of new technologies allowing screening for this defect has permitted new advances in the comprehension of underlying mechanisms. Apart from its implications for host defense, the expression of MPO restricted to myeloid precursors makes MPO mRNA a good marker of acute myeloid leukemia. In addition, during the last few years, involvement of MPO has been described in numerous diseases such as atherosclerosis, lung cancer, Alzheimer's disease and multiple sclerosis. Both strong oxidative activity and MPO genetic polymorphism have been involved. This review summarizes the broad range of diseases involving MPO and points out the possible use of this protein as a new clinical marker and a future therapeutic target.

The present study analyzed the effect of increased concentrations of human apolipoprotein (apo) A-I in transgenic mice serum on paraoxonase activity and on lipid peroxidation. In the transgenic mice serum, in comparison to control (non-transgenic) C57BL/6 mice, we found high concentrations of human apoA-I and high-density lipoprotein (HDL)-cholesterol, but serum lipid peroxidation (basal and free radical-induced) and serum paraoxonase activity were similar in the two mouse groups. Comparing the individual results, no significant correlation was found between free radical-induced serum lipid peroxidation and apoA-I concentrations. Serum paraoxonase activity also did not correlate with serum concentrations of human apoA-I. However, a significant inverse relationship (R 2 =0.75) was observed between the individual values of paraoxonase activity and free radical-induced lipid peroxidation in both mouse groups. Direct analysis of the effect of pure human apoA-I and paraoxonase (using the specific paraoxonase inhibitor PD-92770) on lipid peroxidation also revealed that paraoxonase, but not apoA-I, protects serum lipids from oxidation. We thus conclude that the increased human apoA-I concentration in the mouse serum neither affect serum paraoxonase activity, nor protects against lipid peroxidation, whereas paraoxonase significantly inhibits serum lipid peroxidation.

To determine whether the sialic acid (SA) content of the low-density lipoprotein (LDL) is related to the plasma concentration of autoantibodies to oxidized LDL (oxLDL), we measured the SA content of LDL and the concentrations of oxLDL and autoantibodies to oxLDL in plasma of 20 apparently healthy subjects and 20 patients with advanced coronary atherosclerosis. In the healthy subjects the SA content of LDL correlated positively with plasma concentration of autoantibodies to oxLDL. In agreement with the literature the decreased SA content of LDL was associated with an increased fraction of oxLDL; a decreased fraction of oxLDL was associated with an increased plasma concentration of autoantibodies to oxLDL. In the patients the SA content of LDL and plasma concentrations of oxLDL and autoantibodies to oxLDL were not related. We conclude that the SA content of LDL correlates positively with plasma concentration of autoantibodies to oxLDL in healthy subjects. However, this association may vary depending on the stage of atherogenesis. Although our results suggest dependence of LDL SA content on the clearance of oxidatively modified (desialylated and oxidized) LDL from blood by autoantibodies to oxLDL, the mechanisms regulating the SA content of LDL await further studies.

We have assessed the prevalence of heparin-platelet factor 4 antibodies in a group of 100 patients on chronic intermittent hemodialysis with repeated exposure to heparin in order to establish the clinical significance of an immunological response to heparin. Heparin-induced platelet reactivity was studied in a subgroup of 86 patients. Routine laboratory parameters such as plasma C-reactive protein, albumin and immunoglobulins were included. Clinical endpoints were thrombosis in blood access, change of blood access, other thromboembolic events, bleeding complications, falling platelet count, and ischemic vascular disease or death within 1 year of blood sampling. Blood access thrombosis was a frequent complication (26%), often leading to change of dialysis blood access (21%). Antibodies were detected in 3–6% of hemodialysis patients and antibody levels correlated to blood access thrombosis and change of access (p<0.05). An unexpected finding was that of increased heparin-induced platelet reactivity in 36% of the control group of 24 uremic patients prior to dialysis or exposure to heparin. The findings suggest that antibodies to heparin-platelet factor 4 may contribute to hypercoagulability during hemodialysis, leading to thrombotic complications in affected patients.

In order to diagnose and monitor patients with allergic diseases, we studied parameters which reflect not only the amount of eosinophils but also their state of activation. Morphologic features reveal additional information on the activity state of eosinophils. The number of nuclear lobes, cell size, amount of vacuoles and density of specific granules are considered characteristic features of eosinophils. We conclude that the size of eosinophils is a useful parameter to distinguish patients with and without eosinophilia. The treatment with corticosteroids did not affect morphological characteristics such as the number of vacuoles, granulation density, cell diameter and the nucleus/cell surface ratio, but the number of lobes per nucleus, a marker of eosinophil maturation, decreased significantly.

The availability of long-chain polyunsaturated fatty acids during infancy has been related to neonatal growth and development. Fatty acid concentration at birth is an important predictor of postnatal level. The primary aim of this study was to provide a description of the distribution of n−3 and n−6 polyunsaturated fatty acids in the plasma phospholipid fraction of pregnant women remaining on a Western-style diet and their neonates. The plasma phospholipid polyunsaturated fatty acid composition was determined by gas-liquid chromatography in 889 mother-infant pairs. Blood samples were taken during the first, second and third trimester of pregnancy, at delivery, and from the umbilical vein at birth. Mean (±SD) fatty acid concentrations are reported in mg/l and as percentage of total fatty acids (% wt/wt). In addition, the 10th, 25th, 50th, 75th and 90th percentiles are given. The distribution of docosahexaenoic acid (22:6n−3) and arachidonic acid (20:4n−6) concentrations in umbilical plasma phospholipids is also reported as a function of gestational age and maternal linoleic acid intake during pregnancy. This data can be be used as a reference for future studies and may aid in identifying term infants with a relatively low long-chain polyene status at birth.

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune ® ) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC)-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 μl) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB ® material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray ® source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6–50 μg/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 μg/l in routine use; the instrumentation proved to be reliable and required minimal maintenance.

The aim of this study was to clarify the effects of anti-dsDNA antibodies on the titer and the nuclear staining pattern(s) in a fluorescent antinuclear antibody (FANA) assay using HEp-2 cells. Anti-dsDNA derived from 14 patients with systemic lupus erythematosus (SLE) was individually affinity-purified. The anti-dsDNA titer of the purified anti-dsDNA solution was measured by radioimmunoassay (RIA) or by enzyme-linked immunosorbent assay (ELISA). In the FANA assay, the anti-dsDNA solution was diluted in a stepwise manner and its titer was expressed by the endpoint dilution. The nuclear staining pattern in the anti-dsDNA solution was examined at the 1:5 and 1:20 dilutions and at the endpoint dilution. The anti-dsDNA titers of the affinity-purified anti-dsDNA solution were high enough (13 to 126 IU/ml) to be measured by RIA. However, the antinuclear antibody (ANA) titers of this solution were relatively low: 1:20 to 1:320. In the study of nuclear staining the peripheral pattern was observed in nine of the 14 cases at a 1:5 dilution. However, at the endpoint dilution, all cases exhibited the homogeneous pattern. These findings indicate that in the FANA assay using HEp-2 cells, 1) although serum samples show high anti-dsDNA titers by RIA or by ELISA, the antibodies' direct contribution to ANA titers is limited, and 2) when samples reveal a homogeneous staining pattern at the endpoint dilution, this suggests the presence of anti-dsDNA.

We studied 44 patients with type 1 Gaucher's disease (16 non-treated patients and 28 treated with enzyme replacement therapy). We measured serum levels of chitotriosidase (ChT), neopterin, angiotensin-converting enzyme (ACE), adenosine deaminase (ADA) and β-hexosaminidase (Hex) and its major isoenzymes Hex A and Hex B. In the untreated group of patients, the increase in serum levels was ChT>neopterin>ACE>ADA>Hex, with all decreasing significantly in treated patients (p<0.001). Highly significant correlations were obtained between the markers of monocyte/macrophage activation which were tested (p<0.001). However, partial correlations between serum Hex B (with Hex A constant) and ChT, ACE, neopterin and ADA did not reach statistical significance. This suggests that hepatocytes are the major cellular source of this isoenzyme. Similarly, partial correlation of ChT with neopterin, with the other variables constant, was not significant, which would suggest a different expression of these two markers in Gaucher's disease.

Two new automated, highly sensitive methods for the measurement of C-reactive protein (CRP) were compared using blood samples from active and retired health care workers (116 males, 114 females). The regression equation was y=1.115x−0.0267 for the Kamiya K assay immunoturbidimetric method (x) vs. the Behring BNII nephelometric method (y) with medians (IQR) of 0.156 (0.081 to 0.276) and 0.150 (0.060 to 0.240) mg/dl, respectively. Significant differences in hsCRP values between males and females were observed (p=0.013 and p=0.0018 for Behring and Kamiya methods, respectively). This gender difference in CRP levels was confirmed by analysis of serum samples from 1114 individuals (639 males and 475 females) obtained through Mayo Medical Laboratories reference services, which represents participants from across the United States. The gender difference, which was confirmed (p<0.0001), was independent of age differences as assessed by multiple regression analysis. The observed gender differences have important implications for the establishment of cut-off points for cardiovascular risk stratification.

Serum cholinesterase activities were measured in 300 patients with thermal injuries. The samples were drawn immediately upon admission and thereafter daily until the time of the patients' discharge or death. According to the burn severity a characteristic decrease was noted during the first days. The decline during the first 24 hours as well as its value (measured 24 hours after admission) was found to be correlated with the total body surface area burned. In all patients the decrease in cholinesterase activity persisted for days, but in patients with inhalation trauma this decrease was significantly greater than in patients without lung injury. It seems that serum cholinesterase activity reflects not only the presence of an inhalation trauma. The cholinesterase measurement seems to be also a good method to observe the course of illness after a burn injury. Our data suggest a correlation between cholinesterase activity and morbidity. The inhalation trauma aggravates the thermal trauma.

Psoriasis is characterized by defects in the normal cycle of epidermal development that lead to epidermal hyperproliferation, altered maturation of skin cells, vascular changes and inflammation. Also, psoriasis has been associated with an abnormal plasma lipid metabolism. Changes in plasma lipid and lipoprotein composition in patients with psoriasis may be the reason for the increased risk of atherosclerosis in these patients. We determined serum concentrations of lipids, lipoproteins and apolipoprotein A1 and B (apo A1 and apo B) in 72 patients with psoriasis and 30 agematched controls. Serum lipoprotein (a) (Lp(a)), apo A1 and apo B were measured by immunoprecipitation assays, and the lipids and other biochemical parameters by enzymatic methods. Serum Lp(a) and triglyceride (TG) were significantly higher in patients with psoriasis than in healthy control subjects (p<0.01 for both). Apo B was also found to be higher in the patient group, but the difference was not significant. The levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and apo A1 did not differ significantly from those of the controls. These observations imply that serum Lp(a) and TG concentrations may play a role as risk factors for atherosclerotic disease in patients with psoriasis.

We processed 317 samples from healthy adult volunteers for a complete blood count, including leukocyte differentials and reticulocyte parameters, through five new-generation haematology analysers: Abx Pentra 120 Retic, Coulter Gen-S, Sysmex SE 9500, Abbott Cell Dyn 4000 and Bayer Advia 120. From these data nonparametric 2.5–97.5 percentile reference intervals were calculated for all parameters on all analysers. Some differences were found compared with previously reported reference intervals. Reference intervals for platelet parameters and reticulocytes agreed with these usually accepted. For red blood cell parameters, including haemoglobin and haematocrit, and white blood cell count, including absolute white blood cell differentials, our calculated reference intervals were in agreement with less frequently cited earlier reports, but were lower compared to the usually accepted reference intervals.

This article considers two paintings by the English painter, Joseph Wright of Derby (1734–97), ‘An Experiment on a Bird in the Air Pump’ and ‘The Alchemist in Search of the Philosopher's Stone’ as painterly reflections on the early industrial culture. Particularly interesting in the “Experiment on a Bird” is the broad spectrum of spectator's reactions to the experiment; this can be related to the contemporary debate on the contextualization of science.

HbA 1c is the stable glucose adduct to the N-terminal group of the β-chain of HbA 0 . The measurement of HbA 1c in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA 1c Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the β-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA 1c is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA 1c and HbA 0 are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA 1c routine assays worldwide.