Methods, units and quality requirements for the analysis of haemoglobin A1c in diabetes mellitus.

The formation of glycohemoglobin, especially the hemoglobin A1c (HbA1c) fraction, occurs when glucose becomes coupled with the amino acid valine in the ß-chain of Hb; this reaction is dependent on the plasma concentration of glucose. Since the early 1970s it has been known that diabetics display higher values OF HbA1C because they have elevated blood glucose concentrations. Thus HbA1c has acquired a very important role in the treatment and diagnosis of diabetes mellitus. After the introduction of the first quantitative measurement OF HbA1C, numerous methods for glycohemoglobin have been introduced with different assay principles: From a simple mini-column technique to the very accurate automated high-pressure chromatography and lastly to many automated immunochemical or enzymatic assays. In early days, the results of the quality control reports for HbA1c varied extensively between laboratories, therefore in United States and Canada working groups (WG) of the Diabetes Controls and Complications Trial (DCCT) were set up to standardize the HbA1c assays against the DCCT/National Glycohemoglobin Standardization Program reference method based on liquid chromatography. In the 1990s, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) appointed a new WG to plan a reference preparation and method for the HBA1c measurement. When the reference procedures were established, in 2004 IFCC recommended that all manufacturers for equipment used in HbA1c assays should calibrate their methods to their proposals. This led to an improvement in the coefficient of variation (CV%) associated with the assay. In this review, we describe the glycation of Hb, methods, standardization of the HbA1c assays, analytical problems, problems with the units in which HbA1c values are expressed, reference values, quality control aspects, target requirements for HbA1c, and the relationship of the plasma glucose values to HbA1c concentrations. We also note that the acceptance of the mmol/mol system for HbA1c as recommended by IFCC, i.e., the new unit and reference ranges, are becoming only slowly accepted outside of Europe where it seems that expressing HbA1c values either only in per cent units or with parallel reporting of percent and mmol/mol will continue. We believe that these issues should be resolved in the future and that it would avoid confusion if mmol/mol unit for HbA1c were to gain worldwide acceptance.

Adaptation of the Diazyme Direct Enzymatic HbA1c Assay for a microplate reader at room temperature.

BACKGROUND: Measurement of hemoglobin A(1c) (HbA(1c)) is a key diagnostic criterion and a key parameter for the follow-up of the treatment of diabetes mellitus. Typically, immunochemical assays of HbA(1c) are performed in clinical chemistry analyzers. In this study, we applied the HbA(1c) assay on a microplate reader at room temperature. METHODS: HbA(1c) samples were measured using the Direct Enzymatic HbA(1c) Assay from Diazyme Laboratories (Poway, CA, USA) using a Plate Chameleon Microplate Reader (Hidex Co., Turku, Finland) according to the manufacturer's protocol and a modification of the method to room temperature. The Tosoh G7 HPLC method for HbA(1c) (Tosoh Co., Tokyo, Japan) was used as a comparative method. RESULTS: There was good correlation of HbA(1c) results when the assay was performed at room temperature (+22°C) compared with that at +37°C (r=0.987). The modified method was linear over the HbA(1c) range 4%-14%. Analysis of HbA(1c) results from 50 blood samples by the modified method showed good agreement with the HPLC method (r=0.990). CONCLUSIONS: The modified Diazyme Direct Enzymatic HbA(1c) Assay™ appears to work as good at +22°C as that performed according to manufacturer's protocol at +37°C.

Serum high sensitivity C-reactive protein and cognitive function in elderly women.

BACKGROUND: Inflammation has been linked to cognitive impairment. However, limited data are available on the association between inflammatory markers and cognitive function. OBJECTIVES: We tested the hypothesis that elevated serum concentration of high sensitivity C-reactive protein (hs-CRP), an established marker of low-grade inflammation, predicts cognitive impairment in elderly women. DESIGN: A 12-year population-based follow-up study. PARTICIPANTS: A total of 97 women between 60 and 70 years of age at baseline. METHODS: Serum hs-CRP concentration was measured by a high sensitivity assay. Global cognitive function was measured with the Mini-Mental State Examination (MMSE), and memory and cognitive speed were measured with a detailed cognitive test battery. RESULTS: Higher baseline hs-CRP was associated with poorer memory at 12-year follow-up without adjustment and after adjustment for age, education and depression (standardised regression coefficient beta -0.842, 95% confidence interval -1.602 to -0.083, P = 0.030), and further adjustment for the use of hormone replacement therapy, smoking, serum LDL cholesterol and body mass index (standardised regression coefficient beta -0.817, 95% confidence interval -1.630 to -0.004, P = 0.049). Memory at 12-year follow-up worsened linearly with increasing hs-CRP at baseline (P = 0.048 for linear trend). There was no association between hs-CRP at baseline and cognitive speed or MMSE score at 12-year follow-up. CONCLUSIONS: High serum hs-CRP concentration predicts poorer memory 12 years later in elderly women. Hs-CRP may be a useful biomarker to identify individuals at an increased risk for cognitive decline.

Isolation of drugs from biological fluids by using pH sensitive poly(acrylic acid) grafted poly(vinylidene fluoride) polymer membrane in vitro.

Isolation of acidic and basic model drugs by using pH sensitive poly(acrylic acid) grafted poly(vinylidene fluoride) (PAA-PVDF) cation-exchange membrane from biological fluids was reported. Effects of drug charge and lipophilicity on adsorption were also investigated. In the present study, basic model drugs adsorbed to a considerably greater extent onto the membrane than acidic drugs. Albumin was not adsorbed onto the membrane. Results of our study exposed, that electrostatic interactions between positively charged basic drug and negatively charged PVDF-PAA membrane were the most important factor affecting drug adsorption onto the membrane. Adsorption of acidic and basic drugs onto the PVDF-PAA membrane was not related to drug lipophilicity. The results of present study demonstrated that basic drugs adsorbed extensively onto the membrane, but albumin did not, proposing that PAA-PVDF membrane may be suitable for isolating basic drugs from proteinaceous biological fluids (i.e. serum) for subsequent monitoring and evaluation.

Adsorption of drugs onto a pH responsive poly(N,N-dimethyl aminoethyl methacrylate) grafted anion-exchange membrane in vitro.

The influence of charge and lipophilicity of acidic and basic model drugs on their adsorption onto poly(N,N-dimethyl aminoethyl methacrylic acid) grafted poly(vinylidene fluoride) (DMAEMA-PVDF) membranes was evaluated. The effect of serum proteins (albumin, IgG) and hormones (cortisol, free thyroxine (T(4)F) and thyrotropin (TSH)) on drug adsorption was also studied. Acidic model drugs (antiepileptics and benzodiazepies) adsorbed to a greater extent onto the membrane from Hepes buffer at ionic strength of 25mM and pH 7.0 than basic drugs (antidepressants) did. Adsorption of acidic model drugs was based on electrostatic interactions between positively charged tertiary amino groups of DMAEMA side-chain and acidic negatively charged drug. Albumin diminished the adsorption of drugs from serum onto the membrane. Lipophilicity was related to the adsorption of acidic model drugs from serum onto the membrane. The degree of grafting had the greatest effect on adsorption of lipophilic drugs, but no influence was observed on adsorption of hydrophilic drugs. The present results showed that acidic drugs and albumin adsorbed onto the membrane, which suggests that the PVDF-DMAEMA membrane may be suitable for separating acidic drugs from protein-free substances for subsequent monitoring and evaluation.

Effects of aerobic physical exercise on inflammation and atherosclerosis in men: the DNASCO Study: a six-year randomized, controlled trial.

BACKGROUND: Although regular physical activity is recommended for prevention of cardiovascular diseases, no data are available on its antiatherosclerotic effects in the general population. OBJECTIVE: To determine whether progressive aerobic exercise compared with usual activity slows progression of atherosclerosis in men. DESIGN: A 6-year randomized, controlled trial. SETTING: Eastern Finland. PARTICIPANTS: 140 middle-aged men randomly selected from the population registry. INTERVENTION: Low- to moderate-intensity aerobic exercise. MEASUREMENTS: Atherosclerosis was quantitated ultrasonographically as the mean intima-media thickness in the carotid artery at baseline and at years 2 through 6. RESULTS: On the basis of intention-to-treat analyses, a 19.5% net increase (P < 0.001) in ventilatory aerobic threshold was evident in the exercise group after 6 years. High-sensitivity C-reactive protein levels were statistically nonsignificantly lower in the exercise group than in the control group (P > 0.2). The progression of intima-media thickness in the carotid artery did not differ between the study groups (P > 0.2). A subgroup analysis that excluded men taking statins showed that the 6-year progression of intima-media thickness, adjusted for smoking and annual measures of low-density lipoprotein cholesterol level, systolic blood pressure, and waist circumference, was 40% less in the exercise group (0.12 mm [95% CI, -0.010 to 0.26 mm]) than in the control group (0.20 mm [CI, 0.05 to 0.35 mm]). LIMITATIONS: Only middle-aged white men were included. The intervention included mainly aerobic exercises. CONCLUSIONS: Aerobic physical exercise did not attenuate progression of atherosclerosis, except in a subgroup of men not taking statins.

Interleukin-6 and tumor necrosis factor alpha in relation to myocardial infarct size and collagen formation.

BACKGROUND: Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) levels increase after acute myocardial infarction (AMI) in humans. Experimental data suggest that these cytokines regulate the initiation of scar formation after AMI. We investigated the interrelationships of IL-6 and TNF-alpha, tissue injury, infarct size, cardiac function, and collagen formation in humans. METHODS: Serum and plasma samples were taken on 93 patients receiving thrombolytic treatment for their first AMI. Collagen formation was evaluated by measuring concentrations of serum aminoterminal propeptide of type III procollagen (PIIINP). RESULTS: IL-6 levels increased by 44% (P<.001) and peaked at 24 hours. Peak IL-6 levels correlated positively with area under the curve of creatine kinase MB mass (r=.31, P<.01), peak troponin T level (r=.34, P<.005), and PIIINP measured at discharge (r=.46, P<.001). There were no changes in TNF-alpha levels, and patients with left ventricular dysfunction (EF<40%) had similar TNF-alpha levels as those with preserved left ventricular function. CONCLUSIONS: IL-6 may regulate collagen formation and thus remodeling of the left ventricle after AMI. In addition, TNF-alpha measurement is useless in the assessment of infarct size or left ventricular function during the immediate post-infarction period.

Abnormal hematologic profiles in elite cross-country skiers: blood doping or?

OBJECTIVE: There is widespread public concern about fairness in sports. Blood doping undermines fairness and places athletes' health at risk. The purpose of this study was to examine the prevalence of abnormal hematologic profiles in elite cross-country skiers, which may indicate a high probability of blood doping. SETTING AND PARTICIPANTS: Samples were obtained as part of routine International Ski Federation blood testing procedures from participants at the World Ski Championships. Sixty-eight percent of all skiers and 92% of those finishing in the top 10 places were tested. MAIN OUTCOME MEASURES: Using flow cytometry, we analyzed erythrocyte and reticulocyte indices. Reference values were from the 1989 Nordic Ski World Championships data set and the International Olympic Committee Erythropoietin 2000 project. RESULTS: Of the skiers tested and finishing within the top 50 places in the competitions, 17% had "highly abnormal" hematologic profiles, 19% had "abnormal" values, and 64% were normal. Fifty percent of medal winners and 33% of those finishing from 4th to 10th place had highly abnormal hematologic profiles. In contrast, only 3% of skiers finishing from 41st to 50th place had highly abnormal values. CONCLUSIONS: These data suggest that blood doping is both prevalent and effective in cross-country ski racing, and current testing programs for blood doping are ineffective. It is unlikely that blood doping is less common in other endurance sports. Ramifications of doping affect not only elite athletes who may feel compelled to risk their health but also the general population, particularly young people.

Myoglobin, creatine kinase MB isoforms and creatine kinase MB mass in early diagnosis of myocardial infarction in patients with acute chest pain.

OBJECTIVES: Measurements of myoglobin and creatine kinase (CK)-MB isoforms have been suggested to be sensitive tests for the early diagnosis of myocardial infarction (MI). We have investigated the utility of myoglobin, creatine kinase (CK)-MB isoforms and creatine kinase MB mass (CK-MBm) in early diagnosis of MI using cardiac troponin T (cTnT) positivity as a reference. DESIGN AND METHODS: The study population comprised 440 patients who had had chest pain for less than 12 h. Patients were divided into cTnT negative (cTnT-) or cTnT positive (cTnT+) patients (concentration of cTnT >0.1 microg/L at two different time points during 72 h). RESULTS: At the time of admission to the emergency department receiver operating characteristics (ROC) curves of CK-MB isoforms and CK-MBm were not better than that of myoglobin. Six hours after admission CK-MB isoforms and CK-MBm provided statistically significantly larger areas under the curve (AUC) than myoglobin (p < 0.01). When ROC curves were related to the onset of chest pain (< 3 h, 3-6 h, and > 6 h) there were no significant differences between the cardiac markers studied. CONCLUSIONS: According to the present findings, CK-MB isoforms or myoglobin offer no advantage over CK-MBm as early markers of myocardial infarction.

Normalization of vasoactive changes in preeclampsia precedes clinical recovery.

OBJECTIVE: The objective of this study is to compare the serum levels of fibronectin, nitric oxide (NO), cyclic guanosine-monophosphate, endothelin-1, and 6-keto-prostaglandin-F 1alpha in women with and without preeclampsia before and after delivery. METHODS: We studied 20 singleton pregnancies complicated by preeclampsia, and 20 women undergoing elective cesarean delivery were selected as controls. The normalization of circulating concentrations of maternal plasma NO, cyclic guanosine-monophosphate, fibronectin, endothelin-1, thromboxane-B 2 and renin, and urinary 6-keto-prostaglandin-F 1alpha after delivery was evaluated. RESULTS: Mean systolic and diastolic blood pressure (BP) in the puerperium of preeclamptic women remained high after discharge from hospital, and only circulating fibronectin levels were found to be elevated in affected women at the end of hospital stay 5 days after delivery. Normalization of the imbalance in vasoactive substances and renal impairment in preeclampsia occur more rapidly than the patient's clinical recovery, within 2-3 days postpartum. CONCLUSIONS: Slow normalization of circulating fibronectin concentrations reflects slow recovery of endothelial damage in preeclampsia, which may play a major role in maintaining high BP in the puerperium. Plasma levels of endothelin-1 declined to normal levels by the third postpartum day and the finding is consistent with the hypothesis that endothelin-1 is not the major vasoconstrictor in the pathophysiology of preeclampsia.

The Advia 120 red blood cells and reticulocyte indices are useful in diagnosis of iron-deficiency anemia.

We have investigated the red blood cell (RBC) and reticulocyte indices of the Advia 120 hematology system in assessment of body iron stores as well as in diagnostics of iron-deficiency anemia in two separate study populations. The first study population consisted of a total of 34 apparently healthy females who were found to be anemic (Hb<125gL1) in a screening test. The anemic subjects were classified on the basis of plasma transferrin receptor (TfR) concentration into an iron-deficiency anemia group (TfR concentration 2.4mgL1, n=14) and an adequate iron stores group (TfR concentration <2.4mgL1, n=20). Another study population consisted of 95 hospital patients of whom 31 had depleted iron stores according to TfR concentration. The same population was classified further on the basis of hemoglobin value (Hb<125gL1 for females, Hb<135gL1 for males) into patients with iron-deficiency anemia (n=21) and those with anemia together with adequate iron stores (n=44). In the population of young anemic female students the percentage of hypochromic RBC (%HYPOm) had a remarkably high ROC AUC of 0.98 when evaluating the diagnostic accuracy for the distinction between the patients with iron-deficiency anemia and those with anemia and adequate iron stores. Also, among the hospitalized patients %HYPOm had the highest ROC AUC of 0.77. The diagnostic efficiency provided by the red blood cell and reticulocyte indices was considerably lower in the heterogeneous group of hospitalized patients than in the group of female students. Nevertheless, the advanced RBC and reticulocyte indices may prove to be useful tools in the evaluation of iron status and diagnosis of iron-deficiency anemia.

Serum sialic acid and prostate-specific antigen in differential diagnosis of benign prostate hyperplasia and prostate cancer.

In order to improve the diagnostic accuracy of the serum total and free prostate-specific antigen (PSA) in differential diagnosis between benign prostate hyperplasia (BPH) and prostate cancer, the serum total sialic acid (TSA) was measured and logistic regression (LR) models were built. Significantly higher serum PSA (p<0.001) concentrations were observed in patients with prostate cancer compared to control subjects, but no statistically significant differences were found in serum TSA concentrations between these groups. Serum PSA reliably discriminated patients with prostate cancer from control subjects, the area under the ROC curve (AUC) being 0.991 (0.010). When serum PSA was in the gray zone, from 4 to 10 microg/l, the diagnostic accuracy of PSA in discriminating patients with prostate cancer from BPH patients was very poor, AUC being 0.563 (0.132). However, using the same set of patients the LR model combining serum PSA, free to total PSA ratio and TSA values, as well as digital rectal examination results, had good diagnostic accuracy in discriminating the prostate cancer patients from patients with BPH, the area under the ROC curve being 0.895 (0.054). The present data suggest that the logistic regression model combining laboratory measurements and results of the clinical examination may be a useful adjunct in the differential diagnosis of benign and malignant prostate disease.

Renal effects of uranium in drinking water.

Animal studies and small studies in humans have shown that uranium is nephrotoxic. However, more information about its renal effects in humans following chronic exposure through drinking water is required. We measured uranium concentrations in drinking water and urine in 325 persons who had used drilled wells for drinking water. We measured urine and serum concentrations of calcium, phosphate, glucose, albumin, creatinine, and beta-2-microglobulin to evaluate possible renal effects. The median uranium concentration in drinking water was 28 microg/L (interquartile range 6-135, max. 1,920 microg/L) and in urine 13 ng/mmol creatinine (2-75), resulting in the median daily uranium intake of 39 microg (7-224). Uranium concentration in urine was statistically significantly associated with increased fractional excretion of calcium and phosphate. Increase of uranium in urine by 1 microg/mmol creatinine increased fractional excretion of calcium by 1.5% [95% confidence interval (CI), 0.6-2.3], phosphate by 13% (1.4-25), and glucose excretion by 0.7 micromol/min (-0.4-1.8). Uranium concentrations in drinking water and daily intake of uranium were statistically significantly associated with calcium fractional excretion, but not with phosphate or glucose excretion. Uranium exposure was not associated with creatinine clearance or urinary albumin, which reflect glomerular function. In conclusion, uranium exposure is weakly associated with altered proximal tubulus function without a clear threshold, which suggests that even low uranium concentrations in drinking water can cause nephrotoxic effects. Despite chronic intake of water with high uranium concentration, we observed no effect on glomerular function. The clinical and public health relevance of the findings are not easily established, but our results suggest that the safe concentration of uranium in drinking water may be within the range of the proposed guideline values of 2-30 microg/L.