Adenosine deaminase activity in subjects with normal pregnancy, pregnancy induced hypertension and pre-eclampsia.

BACKGROUND: Both pregnancy and adenosine deaminase (ADA) are associated with depressed cellular mediated immunity. There is little information on ADA activity in pregnant Africans. OBJECTIVE: To determine the serum levels of adenosine deaminase (ADA) in normal pregnancy and pregnancy complicated by hypertension in Nigerian women. METHODS: One hundred and twenty-five pregnant women comprising 35 normal non-pregnant women, 35 normal pregnant women, 35 pregnant women with pregnancy induced hypertension and 20 patients with pre-eclampsia were recruited for the study. Serum adenosine deaminase enzyme (ADA) activity was measured by the Giusti and Galanti spectrophotometric method in all study subjects. RESULTS: The mean serum ADA level in the non-pregnant women was higher than that in the normal pregnant women (23.21 +/- 6.3 v 14.69 +/- 3.2, p<0.001). Amongst the pregnant women, mean serum ADA in the hypertensive and pre-eclamptic women was significantly higher than that in the normal pregnant group (p<0.001). CONCLUSION: These findings indicate a probable decrease in cellular immunity in normal pregnancy and an enhanced cell mediated immunity in pre-eclampsia.

Plasma total and ultrafiltrable calcium in normal pregnancy, hypertension in pregnancy and pre-eclampsia.

OBJECTIVE: To measure the ultrafiltrable and total plasma calcium in normal pregnancy and pregnancies complicated with hypertension and pre-eclampsia. PATIENTS AND METHODS: Total and ultrafiltrable calcium concentrations were measured in maternal plasma from non-pregnant (35), normal pregnant (35), Pregnancy induced hypertension (35) and pre-eclamptic (20) women. Plasma total calcium level was measured by the o'cresolphthalein method. Ultrafiltrate of plasma was obtained using the Amicon MPS-1 micro-partition device. RESULTS: There was no significant difference in the plasma total calcium level between the non- pregnant group and the pregnant group (normal, hypertensive and pre-eclamptic). However there was a significant reduction in the ultrafiltrable (protein free and complexed) calcium level in the pregnant group compared to the non-pregnant group (1.15mmol/L +/- 0.23 Vs 1.25mmol/L +/- 0.13) p<0.05. CONCLUSION: Measurement of the ultrafiltrable calcium in addition to total calcium assay may be more useful in assessing calcium status in normal and complicated pregnancies.

Analytical evaluation of HgbA1c, microalbumin, CRP, and RF on Architect ci8200 integrated system and workflow performance evaluation using computer simulation.

BACKGROUND: Recently, hemoglobin A1c (HgbA1c), microalbumin (MA), C-reactive protein (CRP) and rheumatoid factor (RF) have been introduced on high throughput general chemistry system. We evaluated analytical performance of these assays on an integrated clinical chemistry and immunoassay analyzer and studied the impact of testing these assays on these systems on the overall efficiency of the analyzer, via computer simulation. METHODS: The analytical performance was measured by determining precision, linearity and correlation of patient sample results with in-house testing methodology. MedModel simulation software is used to develop simulation model and process efficiency is determined by measuring turnaround times and resource utilization. RESULTS: Between-days CVs ranged from 8.59% for MA to 3.22% for HgbA1c level 1 controls. Less than 2% carryover for all 4 methods was observed on the integrated analyzer. For HgbA1c on HPLC analyzer, the minimum and maximum TAT for a batch of 50 samples was 3.78 and 160 min, respectively, while for the integrated system it was 28.2 and 35.1 min, respectively. Labor utilization for the 2 processes ranged from 3.21% to 3.75%. CONCLUSION: Chemistry module on an integrated system can be used to determine the HgbA1c and other serum proteins.

Nuclear deoxyribonuclease activities in human lymphoblastoid and mouse melanoma cells. A comparative study.

Deoxyribonuclease activities were examined in isoelectric focusing fractions of non-histone, chromatin-associated and nucleoplasmic proteins of isolated normal human lymphoblastoid and mouse melanoma cell nuclei using parallel procedures. A very similar series of eight DNA endonucleases, each active on calf thymus DNA and containing no exonuclease activity, were found in the chromatin proteins of both cell lines. Several differences were observed: an activity in human cells at pI 6.6 was absent from murine cells, and there was an increased activity in mouse cells at pI 4.4 and a decreased activity at pI 7.3, as compared with corresponding human cell activities. Assay of these fractions against supercoiled, circular phage PM2 DNA showed greater activity among the fractions with acidic pI valves and slightly lower activities in the murine cells than in the human cells. Analysis of the nucleoplasmic fractions showed a series of DNA endonuclease and exonuclease activities which were again very similar between the two cell lines, although greater endonuclease activity at pI 4.4 occurred in mouse than in human nucleoplasm. These results demonstrate an entire series of deoxyribonuclease activities in both chromatin and nucleoplasm which are nearly identical in two very different mammalian cell lines, suggesting that many of these enzymes are ubiquitous in mammalian cell nuclei.

Abnormal expression of genes associated with development and inflammation in the heart of mouse maternal phenylketonuria offspring.

This study describes gene expression in the fetus hearts obtained from mouse model for phenylketonuria. These hearts have cardiovascular disease (CVD). Therefore genes involved in CVD were examined. Several genes associated with heart development and inflammation were found to be altered. In order to investigate whether the abnormal gene expression alters transcription and translation, the levels of troponin mRNA and protein were determined. One step real time RT-PCR showed a reduction in cardiac troponin I, troponin T2 and ryanodine receptor 2. Determination of troponin I and T protein levels showed reduced levels of these proteins. Our results suggest that altered gene expression affects protein production. These changes are likely involved in the cardiovascular defects seen in the mouse.

Serum interleukin-6 in bacterial and nonbacterial acute otitis media.

BACKGROUND: Increasing prevalence of antibiotic-resistant bacteria is a serious clinical problem that calls for reduction of unnecessary use of antibiotics. Acute otitis media (AOM) is the most common reason for antibiotic therapy in the United States. Approximately 30% of AOM cases do not have a bacterial etiology. Rapid identification of these cases could help withhold unnecessary antibiotic treatment. OBJECTIVE: To determine the usefulness of serum levels of interleukin-6 (IL-6), an acute phase cytokine shown to be a reliable marker of neonatal bacterial infection, in differentiation between bacterial and nonbacterial AOM in children. STUDY DESIGN: IL-6 was measured in stored serum samples from 184 children (mean age, 22 months) with AOM who were enrolled in antibiotic efficacy trials at our department. The samples were obtained at enrollment and at 9 to 12 days after initiation of antibiotic therapy. Sera from 21 uninfected children (mean age, 23 months) were used as controls. The etiology of AOM was determined by bacterial and viral cultures as well as respiratory syncytial virus antigen detection in the middle ear fluids obtained by tympanocentesis. RESULTS: Bacterial etiology of AOM was confirmed in 125 children (68%), whereas in 59 children (32%) no bacterial pathogen could be detected in the middle ear fluid. Children with bacterial AOM had significantly higher IL-6 levels than those with nonbacterial AOM (median, 11.5 vs 3.7 pg/mL). However, this difference was almost entirely attributable to pneumococcal AOM specifically. IL-6 levels in children with AOM caused by Streptococcus pneumoniae were significantly higher (median, 40.1 pg/mL) than in AOM caused by Haemophilus influenzae (7.3 pg/mL) or Moraxella catarrhalis (6.8 pg/mL). At the cutoff value of 30 pg/mL, the specificity of IL-6 for detection of pneumococcal AOM was 91% with a sensitivity of 61%, but its sensitivity for detection of bacterial AOM in general was only 27%. CONCLUSIONS: Low levels of IL-6 do not rule out bacterial etiology of AOM in general; therefore, IL-6 is not sensitive enough as a marker of bacterial AOM. Surprisingly, serum IL-6 levels in pneumococcal AOM were significantly higher than the levels associated with other bacterial AOM, and serum IL-6 levels of >30 pg/mL were highly specific for pneumococcal AOM. These findings suggest a distinctive role for S pneumoniae in the pathogenesis of AOM.

Evaluation of immuno-1 toxoplasma IgG assay in the prenatal screening of toxoplasmosis.

We evaluated the Immuno-1 (Bayer Diagnostics, Tarrytown, New York, USA) and IMx (Abbott Laboratories, Chicago, Illinois, USA) toxoplasma IgG assays in 298 (223 fresh in-house prenatal + 75 supplied by Bayer) specimens over 15 days. Discordant results were resolved by indirect fluorescence assay (Gull Laboratories, Salt Lake City, Utah, USA). The performance of Immuno-1 assay was found to be comparable to the IMx assay. Immuno-1, being a random access analyzer with minimum hands-on time requirements may have an advantage in the overall laboratory efficiency.

Prospective study of IgM to Toxoplasma gondii on Beckman Coulter's Access(TM) immunoassay system and comparison with Zeus ELISA and gull IFA assays.

We compared a new assay for Toxoplasma IgM on the Access analyzer (Beckman Coulter, Inc., Chaska, MN, USA), a random access instrument based on the principle of paramagnetic particle enzyme immunoassay with an enzyme-linked immunosorbent assay (ELISA) (Zeus Scientific, Inc., Raritan, NJ, USA) and an immunofluorescent assay (IFA) (Gull Laboratories, Inc., Salt Lake City, UT, USA). Four hundred fresh, unfrozen clinical samples from pregnant women (n = 154), HIV positive patients (n = 41), and patients in whom infection with Toxoplasma gondii was suspected (n = 200) were collected and assayed over a three month period. The specificity of the Access assay was compared to the consensus results. Results that were discrepant between the ELISA and IFA were resolved using a third IFA (Zeus). Once resolved, the specificity for the Access assay, the Zeus ELISA and the Gull IFA were 99.22%, 97.91%, and 99.45%, respectively. We conclude that the Access assay specificity is comparable to consensus results, minimizing false positive results; and because it is a random access instrument, it may be preferable over batch methods.

Ionized magnesium in the homeostasis of cells: intracellular threshold for Mg(2+) in human platelets.

The role of extracellular magnesium ions in the homeostasis of intracellular ionized magnesium ([Mg(2+)](i)) in human platelets was studied. For media containing 0.00 to 0.60 mmol/l of extracellular ionized magnesium ([Mg(2+)](o)), the mean [Mg(2+)](i) fluctuated between 533 and 760 micromol/l. As the [Mg(2+)](o) was increased to 1.5 mmol/l, the [Mg(2+)](i) increased proportionately and peaked at 1470.1 micromol/l. Additional increase in the [Mg(2+)](o) from 1.50 to 6.00 mmol/l resulted in decreased [Mg(2+)](i) until it equilibrated between 739 and 776 micromol/l. The influx of Mg(2+) at [Mg(2+)](o) of 0.60 and 1.50 mmol/l was studied using verapamil, a calcium channel inhibitor, and ouabain, an inhibitor of the Na/K pump, respectively. The verapamil (25 mmol/l) blocking experiments resulted in a 92.4% inhibition of the Mg(2+) influx into the platelet at a [Mg(2+)](o) of 1.50 mmol/l. Ouabain (0.5 and 2.5 mmol/l) showed an enhancement effect on the influx of Mg(2+) at [Mg(2+)](o) of 0.60 mmol/l and no effect at 1.50 mmol/l. The effect of verapamil indicates that ion channels that are homologous to calcium ion channels may be involved in the influx of Mg(2+) into the platelets. The inhibition of Mg(2+) influx for [Mg(2+)](o) greater than 1.50 mmol/l may illustrate a protective mechanism that attempts to maintain the viability of platelets at abnormally high [Mg(2+)](o). These results suggest that there is an intracellular Mg(2+) threshold of 1500 micromol/l, above which an active mechanism prevents further influx of Mg(2+).

Accuracy and precision of point-of-care testing for glucose and prothrombin time at the critical care units.

The use of point-of-care testing (POCT) in critical care patient units has continued to increase since the 1980s. This increase is due to the need for prompt therapeutic interventions that may impact mortality and morbidity, and reduce the overall cost of healthcare for critically ill patients. The diagnostic manufacturing industry has risen to this challenge by introducing portable and/or handheld analyzers for use at the point-of-care. In order to ensure the public safety in the USA, the Food and Drug Administration (FDA) must approve the use of each POCT analyzer. The FDA approval is based on established performance criteria that includes relative accuracy and precision documentation. This study evaluated the precision and accuracy of the POCT prothrombin time and glucose analyzers relative to the manufacturers' specifications, to the internal QC in the main laboratory, and to the results of the external proficiency-testing program. The QC for the prothrombin time had a precision that ranged from 2.84% to 3.45% (POCT) and from 1.27-1.66% (main laboratory). The precision for the glucose QC ranged from 5% to 5.2% (POCT) and 0.9-2.7% (main laboratory). Using the results of the external proficiency testing, the inter-laboratory CV% for the POCT prothrombin time ranged from 3.5% to 5.0% and the main laboratory had a range of 2.5-2.9%. The inter-laboratory CV% ranges for glucose POCT and the main laboratory were 4.9-10.6% and 1.8-3.5%, respectively. The main laboratory analyzers proved to be more accurate than the POCT analyzers as indicated by comparison to the mean prothrombin time and glucose results of all participating laboratories in the proficiency testing program.

Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions.

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4 degrees C, or frozen at -20 degrees C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4 degrees C.

Apparatus and method for interfacing capillary electrophoresis and chemiluminescence nitrogen detection for the analysis of nitrogen-containing compounds.

We have developed an interface that allows the specific detection of nitrogen-containing compounds by using a chemiluminescence nitrogen detector. The feasibility of using this interface was demonstrated by separating and detecting two nitrogen-containing compounds, p-aminosalicylic acid and L-phenylalanine. Although baseline separation was achieved, the theoretical plates were lower when compared to UV detection (25000 vs. approximately 85000). A sensitivity of 75 ng (approximately 500 pmol) per injection was achieved with this system which is adequate for pharmaceutical and biotech applications.

Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells.

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated DNA.

Intracellular ionized calcium and increasing doses of lithium chloride therapy in healthy Sprague-Dawley rats.

The use of lithium salts in the prophylaxis and treatment of several psychiatric and neurologic disorders continues to be well accepted despite the apparent lack of understanding regarding its mode of action at the molecular level. This lack of delineation in the mechanism of action is supported by numerous conflicting publications. Despite the lack of understanding, a role for calcium in the manifestation of lithium's action is a constant singular consensus. Intracellular ionized calcium ([Ca2++]i) is involved in the proper functioning of cells because of its role in the second messenger pathway. It is therefore essential to evaluate the effect of lithium on intracellular calcium metabolism in a well-defined system. In this study, platelets loaded with Fura-2-Acetoxymethyl were used to evaluate the effect of intraperitoneally administered lithium chloride at 0, 2.5, 5.0, 7.5, and 10 mmol/kg body wt. on [Ca++]i. The results showed a slight relative increase in serum Ca++ that correlated well with the dose of LiCl administered to the rats. The baseline [Ca++]i were comparable in the study groups, but the response to thrombin stimulation was more pronounced at LiCl doses of 2.5, 5.0, and 7.5/kg body wt. compared with control and rats treated with 10 mmol LiCl/kg body wt. This finding suggests a dose-dependent response of [Ca++]i to LiCl treatment. The observation may therefore explain the variations that have been reported in [Ca++]i studies with respect to LiCl therapy using different doses.

Serum ionized magnesium changes during and immediately after liver transplantation.

Alterations in magnesium (Mg) homeostasis during and after orthotopic liver transplantation are common. The purpose of this study is to compare total Mg (TMg), calculated ionized Mg (cMg++) and measured ionized Mg (mMg++) during and immediately following liver transplantation. The newly developed first generation ion selective electrode analyzer, AVL 988-4, was used to measure mMg++ in 63 serum samples from 3 transplant recipients and 48 serum samples from 48 healthy volunteers. Analysis was divided into intraoperative (stages 1 to 3) and postoperative periods. Decreased TMg, cMg++ and mMg++ levels were observed intraoperatively and > 2 weeks postoperatively. The cMg++ levels were consistently higher than mMg++, presumably owing to the fact that the equation used for the calculation does not take complex-Mg++ into account. A better correlation was observed between mMg++ and cMg++ in the transplant group (r = 0.87 to 0.99) compared to controls (r = 0.74). The usefulness of direct measurement of Mg++ in liver transplantation remains to be determined.

Intracellular calcium and hydrogen ions in diabetes mellitus.

Diabetes mellitus is a multi-component syndrome that is often complicated by angiopathy which is partly due to enhanced platelet functions. Using fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2 AM, changes was evaluated in the concentration of baseline and thrombin-stimulated increases in intracellular ionized calcium (Ca2+i) relative to hydrogen ions in the platelets from control, insulin-treated, and non-treated diabetic rats. The cytosol of platelets from the diabetic rats were more acidic compared to the insulin-treated and normal control rats. The increased intracellular hydrogen ion concentration [H+] or decreased pH (pH) in the diabetic rat platelets is associated with an increased baseline [Ca2+]i. Upon stimulation with thrombin, the mean peak [Ca2+]i for the insulin-treated (309 +/- 97 nmol/L) and untreated (339 +/- 135 nmol/L) diabetic rats was significantly higher than the concentration for the normal rats (213 +/- 101 nmol/L). Treatment with insulin attempts to correct the diabetes-induced elevation in the baseline of [Ca2+]i and intracellular H+. These results suggest that the relationships between Ca2+ and H+ relative to binding sites are similar in the intra- and extracellular compartments. It is our conclusion that the enhanced platelet activity and associated development of vascular diseases in diabetes may be due to an increased intracellular H+ that caused an increased baseline [Ca2+]i in diabetes mellitus.

Particles internalization, oxidative stress, apoptosis and pro-inflammatory cytokines in alveolar macrophages exposed to cement dust.

Exposure to cement dust is one of the most common occupational dust exposures worldwide, but the mechanism of toxicity has not been fully elucidated. Cement dust (N) and clinker (C) samples collected from Nigeria and another sample of cement dust (U) collected from USA were evaluated using alveolar macrophage (NR8383) cell culture to determine the contribution of different sources of cement dust in the severity of cement dust toxicity. Cement dust particles internalization and morphologic alterations using transmission electron microscopy (TEM), cytotoxicity, apoptotic cells induction, intracellular reactive oxygen species generation, glutathione reduction, TNF-α, IL-1ß, and CINC-3 secretion in alveolar macrophages (NR8383) exposed to cement dust and clinker samples were determined. Particles were internalized into the cytoplasmic vacuoles, with cells exposed to U showing increased cell membrane blebbing. Also, NR8383 exposed to U show more significant ROS generation, apoptotic cells induction and decreased glutathione. Interleukin-1ß and TNF-α secretion were significantly more in cells exposed to both cement dust samples compared with clinker, while CINC-3 secretion was significantly more in cells exposed to clinker (p < 0.05). Endocytosis, oxidative stress induced-apoptosis and induction of pro-inflammatory cytokines may be key mechanisms of cement dust immunotoxicity in the lung and toxicity may be factory dependent.

Lead, mercury, cadmium, chromium, nickel, copper, zinc, calcium, iron, manganese and chromium (VI) levels in Nigeria and United States of America cement dust.

This study was aimed at investigating the relative abundance of heavy metals in cement dust from different cement dust factories in order to predict their possible roles in the severity of cement dust toxicity. The concentrations of total mercury (Hg), copper (Cu), chromium (Cr), cadmium (Cd), nickel (Ni), manganese (Mn), lead (Pb), iron (Fe) and chromium (VI) (Cr (VI)) levels in cement dust and clinker samples from Nigeria and cement dust sample from the United States of America (USA) were determined using graphite furnace atomic absorption (GFAAS), while Zn and Ca were measured by flame atomic absorption spectrophotometry (FAAS), and Cr (VI) by colorimetric method. Total Cu, Ni and Mn were significantly higher in cement dust sample from USA (p<0.05), also, both total Cr and Cr (VI) were 5.4-26 folds higher in USA cement dust compared with Nigeria cement dust or clinker (p<0.001). Total Cd was higher in both Nigeria cement dust and clinker (p<0.05 and p<0.001), respectively. Mercury was more in both Nigeria cement dust and clinker (p<0.05), while Pb was only significantly higher in clinker from Nigeria (p<0.001). These results show that cement dust contain mixture of metals that are known human carcinogens and also have been implicated in other debilitating health conditions. Additionally, it revealed that metal content concentrations are factory dependent. This study appears to indicate the need for additional human studies relating the toxicity of these metals and their health impacts on cement factory workers.

The potential benefits and adverse effects of phytic Acid supplement in streptozotocin-induced diabetic rats.

In this study, the effect of phytic acid supplement on streptozotocin-induced diabetic rats was investigated. Diabetic rats were fed rodent chow with or without phytic acid supplementation for thirty days. Blood and organ samples were collected for assays. The average food intake was the highest and the body weight gain was the lowest in the group fed phytic acid supplement compared to the diabetic and normal control groups. There was a downward trend in intestinal amylase activity in the group fed phytic acid supplement compared to the other groups. The spike in random blood glucose was the lowest in the same group. We noted reduced serum triglycerides and increased total cholesterol and HDL cholesterol levels in the group fed phytic acid supplement. Serum alkaline phosphatase and alanine amino transferase activities were significantly (P < 0.05) increased by phytic acid supplementation. Systemic IL-1 ß level was significantly (P < 0.05) elevated in the diabetic control and supplement treated groups. The liver lipogenic enzyme activities were not significantly altered among the groups. These results suggest that phytic acid supplementation may be beneficial in the management of diabetes mellitus. The observed adverse effect on the liver may be due to the combined effect of streptozotocin-induced diabetes and phytic acid supplementation.