European Biological Variation Study (EuBIVAS): within- and between-subject biological variation estimates of ß-isomerized C-terminal telopeptide of type I collagen (ß-CTX), N-terminal propeptide of type I collagen (PINP), osteocalcin, intact fibroblast growth factor 23 and uncarboxylated-unphosphorylated matrix-Gla protein-a cooperation between the EFLM Working Group on Biological Variation and the International Osteoporosis Foundation-International Federation of Clinical Chemistry Committee on Bone Metabolism.

We have calculated the biological variation (BV) of different bone metabolism biomarkers on a large, well-described cohort of subjects. BV is important to calculate reference change value (or least significant change) which allows evaluating if the difference observed between two consecutive measurements in a patient is biologically significant or not. INTRODUCTION: Within-subject (CVI) and between-subject (CVG) biological variation (BV) estimates are essential in determining both analytical performance specifications (APS) and reference change values (RCV). Previously published estimates of BV for bone metabolism biomarkers are generally not compliant with the most up-to-date quality criteria for BV studies. We calculated the BV and RCV for different bone metabolism markers, namely ß-isomerized C-terminal telopeptide of type I collagen (ß-CTX), N-terminal propeptide of type I collagen (PINP), osteocalcin (OC), intact fibroblast growth factor 23 (iFGF-23), and uncarboxylated-unphosphorylated Matrix-Gla Protein (uCuP-MGP) using samples from the European Biological Variation Study (EuBIVAS). METHODS: In the EuBIVAS, 91 subjects were recruited from six European laboratories. Fasting blood samples were obtained weekly for ten consecutive weeks. The samples were run in duplicate on IDS iSYS or DiaSorin Liaison instruments. The results were subjected to outlier and variance homogeneity analysis before CV-ANOVA was used to obtain the BV estimates. RESULTS: We found no effect of gender upon the CVI estimates. The following CVI estimates with 95% confidence intervals (95% CI) were obtained: ß-CTX 15.1% (14.4-16.0%), PINP 8.8% (8.4-9.3%), OC 8.9% (8.5-9.4%), iFGF23 13.9% (13.2-14.7%), and uCuP-MGP 6.9% (6.1-7.3%). CONCLUSIONS: The EuBIVAS has provided updated BV estimates for bone markers, including iFGF23, which have not been previously published, facilitating the improved follow-up of patients being treated for metabolic bone disease.

Experimental study showed that adding fortifier and extra-hydrolysed proteins to preterm infant mothers' milk increased osmolality.

AIM: Measuring milk osmolality after adjustable fortification is clinically relevant, as values exceeding recommended safety thresholds might result in gastrointestinal consequences. The aim of this study was to evaluate the effect of four fortification levels and storage time on the osmolality of human milk. METHODS: This was an experimental study using 71 spare samples of fresh breastmilk collected from 31 mothers of preterm infants. Osmolality was measured before and after adding commercial human milk fortifier containing dextrinomaltose and hydrolysed proteins at four different concentrations. Measurements were performed at various points during the 23 hours after fortification. RESULTS: The mean basal osmolality of the 71 human milk samples was 296 ± 14 milliosmoles (mOsm)/kg, and these remained stable over a period of 23 hours. Just after fortification, the four fortified formulas showed higher osmolalities than the nonfortified human milk, ranging between 384 ± 14 and 486 ± 15 mOsm/kg, respectively (p < 0.01). This osmolality increased significantly from 20 minutes to 23 hours after fortification (p < 0.05). CONCLUSION: Adding fortifier and extra-hydrolysed proteins to human preterm milk increased osmolality, and these osmolality levels also increased with time. We recommend evaluating the risk of hyperosmolality when a higher fortification level is needed, to avoid gastrointestinal problems.

Systematic review of the biological variation data for diabetes related analytes.

BACKGROUND: Objective interpretation of laboratory test results used to diagnose and monitor diabetes mellitus in part requires the application of biological variation data (BVD). The quality of published BVD has been questioned. The aim of this study was to quality assess publications reporting BVD for diabetes-related analytes using the Biological Variation Data Critical Appraisal Checklist (BIVAC); to assess whether published BVD are fit for purpose and whether the study design and population attributes influence BVD estimates and to undertake a meta-analysis of the BVD from BIVAC-assessed publications. METHODS: Publications reporting data for glucose, HbA1c, adiponectin, C-peptide, fructosamine, insulin like growth factor 1 (IGF-1), insulin like growth factor binding protein 3 (IGFBP-3), insulin, lactate and pyruvate were identified using a systematic literature search. These publications were assessed using the BIVAC, receiving grades A, B, C or D, where A is of highest quality. A meta-analysis of the BVD from the assessed studies utilised weightings based upon BIVAC grades and the width of the data confidence intervals to generate global BVD estimates. RESULTS: BIVAC assessment of 47 publications delivered 1 A, 3 B, 39C and 4 D gradings. Publications relating to adiponectin, C-peptide, IGF-1, IGFBP-3, lactate and pyruvate were all assessed as grade C. Meta-analysis enabled global BV estimates for all analytes except pyruvate, lactate and fructosamine. CONCLUSIONS: This study delivers updated and evidence-based BV estimates for diabetes-related analytes. There remains a need for delivery of new high-quality BV studies for several clinically important analytes.

Serum levels of alpha-tocopherol (vitamin E) in Parkinson's disease.

To elucidate the possible role of vitamin E in the pathogenesis of Parkinson's disease (PD), we compared serum levels of alpha-tocopherol (vitamin E), measured by high-performance liquid chromatography, and the vitamin E/cholesterol ratio of 42 Parkinson's disease (PD) patients using their spouses as the control group. The serum levels of vitamin E did not differ significantly between the groups (13.84 +/- 0.56 micrograms/ml for PD and 14.80 +/- 0.57 micrograms/ml for controls), nor did the vitamin E/cholesterol ratio (0.64 +/- 0.03 for both groups). There was no influence of antiparkinsonian therapy on vitamin E or the vitamin E/cholesterol ratio. Serum levels of the vitamin E and vitamin E/cholesterol ratio did not correlate with age, age at onset, scores of the Unified Parkinson's Disease Rating Scale or the Hoehn and Yahr staging in the PD group. These results suggest that serum vitamin E concentrations do not play a role in the pathogenesis of PD.

Serum levels of beta-carotene and other carotenoids in Parkinson's disease.

To elucidate the possible role of carotenoids in the risk for developing Parkinson's disease (PD), we compared serum levels of beta-carotene, alpha-carotene and lycopene, measured by high performance liquid chromatography, of 61 PD patients using their spouses as the control group. The serum levels of these 3 carotenoids did not differ significantly between PD patients and control groups. There was no influence of antiparkinsonian therapy on serum carotenoids levels, and these did not correlate with age, age at onset, scores of the Unified Parkinson Disease Rating Scale or the Hoehn and Yahr staging in the PD group. These results show that serum carotenoids concentrations are apparently unrelated to the risk for developing PD.

Serum lipid peroxides in patients with Parkinson's disease.

To elucidate whether high serum lipid peroxidation rates may increase the risk of developing Parkinson's disease (PD), we assessed serum levels of malondialdehyde (MDA), an intermediate in lipid peroxidation processes, in 37 PD patients, with their spouses as the control group. Serum MDA levels did not differ significantly between these two groups (8.7 +/- 0.51 and 8.8 +/- 0.48 nmol/ml, resp.), and were not influenced by antiparkinsonian therapy in the PD patients. Serum MDA levels were inversely correlated with age and age at onset (P less than 0.01) in the PD group, but they were not correlated with disease duration, Unified Parkinson's Disease Rating Scale scores or Hoehn and Yahr staging. In the control group there was no correlation between serum MDA and age. These results suggest that, although serum levels of lipid peroxides were similar in both the PD and control groups, high serum lipid peroxidation rates might constitute a risk factor for younger onset of PD in predisposed individuals.

Serum levels of ascorbic acid (vitamin C) in patients with Parkinson's disease.

To elucidate the possible role of vitamin C in the risk for developing Parkinson's disease (PD), we compared serum levels of ascorbic acid (vitamin C), measured by a fluorometric method, of 63 PD patients using their spouses as the control group. The serum levels of vitamin C did not differ significantly between the groups (47.13 +/- 0.89 micrograms/ml for PD and 47.60 +/- 0.60 micrograms/ml for controls). There was no influence of antiparkinsonian therapy on vitamin C. Serum levels of vitamin C did not correlate with age, age at onset and duration of the disease, scores of the Unified PD Rating Scale or the Hoehn and Yahr staging in the PD group. These results suggest that serum vitamin C concentrations are apparently unrelated to the risk of developing PD.

Serum levels of zinc and copper in patients with Parkinson's disease.

Several recent studies have shown decreased copper and increased zinc concentrations in the substantia nigra and increased copper concentrations in the cerebrospinal fluid of Parkinson's disease patients. To elucidate whether changes in serum levels of these trace elements may increase the risk of developing Parkinson's disease (PD), we assessed serum levels of zinc and copper by flame atomic absorption spectrophotometry, and albumin and ceruloplasmin, in 32 (Zn) and 39 PD patients (Cu), respectively, with their spouses as the control group. Serum zinc, albumin, copper and ceruloplasmin levels and the zinc/albumin and copper/ceruloplasmin ratios, did not differ significantly between the two groups and were not influenced by antiparkinsonian therapy in the PD patients. Serum zinc/albumin ratio (r = 0.43), ceruloplasmin (r = -0.36) and copper/ceruloplasmin ratio (r = 0.36) correlated significantly with age, but not with age of onset, duration of the disease, scores of the Unified Parkinson's Disease Rating Scale and Hoehn and Yahr staging in PD patients. These values did not correlate with age in the control group. These results suggest that serum levels of zinc and copper do not play any role as risk factors for PD.

Serum levels of vitamin A in Parkinson's disease.

To elucidate a possible role of vitamin A in the pathogenesis of Parkinson's disease (PD) we compared serum levels of retinol (vitamin A), measured by HPLC, and the vitamin A/retinol binding protein (RBP) ratio of 42 PD patients (22 males and 20 females, mean age 67.3 +/- 1.34 years) and their respective spouses as control group (20 males and 22 females, mean age 66.2 +/- 1.42). The serum levels of vitamin A did not differ significantly between the 2 groups (0.59 +/- 0.03 microgram/dl for PD patients and 0.57 +/- 0.03 microgram/dl for controls), nor did the vitamin A/RBP ratio (0.87 +/- 0.04 and 0.82 +/- 0.03, respectively). There was no influence of antiparkinsonian therapy on vitamin A or vitamin A/RBP ratio. Serum levels of vitamin A, and vitamin A/RBP ratio did not correlate with age, age at onset, scores of the Unified Parkinson's Disease Rating Scale or the Hoehn and Yahr staging in the PD group. These results suggest that serum concentrations of vitamin A, do not play a role in the pathogenesis of PD.

Peripheral iron metabolism in patients with Parkinson's disease.

To elucidate the possible role of peripheral metabolism of iron in the risk for developing Parkinson's disease (PD), we compared serum levels of iron, transferrin and ferritin, and 24-h iron excretion in urine after a single intramuscular dose of 1 mg/kg desferrioxamine, in 68 PD patients and their spouses as the control group. All these values did not differ significantly between the groups, they were not influenced by antiparkinsonian therapy, and they did not correlate with age, age at onset and duration of the disease, scores of the Unified PD Rating Scale or the Hoehn and Yahr staging in the PD group, with the exception of the 24-h urinary iron excretion with the duration of the disease (r = 0.32, p < 0.05). These results suggest that peripheral metabolism of iron is apparently unrelated to the risk of developing PD.

Plasma levels of nitrates in patients with Parkinson's disease.

It has been suggested that nitric oxide (NO) could be implicated in the neuronal degeneration of substantia nigra compacta in patients with Parkinson's disease (PD). To ascertain the possible role of NO as risk factor for PD, we studied the plasma levels of nitrate (oxidation product that provides an indirect estimation of NO), in 68 PD patients and 68 matched-controls. The plasma levels of nitrate did not differ significantly between PD patient and control groups (44.5 +/- 2.46 and 44.8 +/- 2.67 mumol/l, respectively). They were not influenced by antiparkinsonian drug and they did not correlate with age at onset, duration, scores of the Unified Parkinson's Disease Rating scales and Hoehn and Yahr staging in the PD group. These data suggest that plasma levels of nitrate are apparently unrelated to the risk for PD.

[A simultaneous study of intestinal permeability and the test of H2 in the expired breath of celiac children]. / Estudio simultáneo de la permeabilidad intestinal y la prueba de H2 en aire espirado en niños celíacos.

Permeability test with sugars (PTS) as a non-invasive diagnostic tool is of special interest in pediatric patients. This study evaluated the relationship between PTS and antigliadin antibodies (AA) with the intestinal biopsy and studied whether an altered expired hydrogen test (EHT) could interfere in this relation in celiac patients. Thirty children (13 girls, 17 boys) with diagnosis of celiac disease (ESPGAN criteria) were divided into three groups according to this histopathology: Group A (n = 23) normal biopsy; Group B (n = 7) biopsy grade III or IV; Group C (n = 25) controls. The intestinal permeability test showed significative differences (p < 0.01) of Group B (0.159 +/- 0.03) with Group A (0.048 +/- 0.005) and with the control Group C (0.039 +/- 0.002). The cut-off for the Youden maximum index (0.75) was 0.092 and ROC SE 0.87 +/- 0.7. The results of the AA were related to those of the PTS. The variance analysis showed that the results of the expired hydrogen test do not interfere statistically on the good correlation of PTS with the histology. The PTS is a good indicator of the status of intestinal mucosa. There is a good correlation between PTS and AA in celiac children. A pathological expired hydrogen test seems not to interfere with the correlation found between PTS and histological damage in our series.

External quality assurance programs as a tool for verifying standardization of measurement procedures: Pilot collaboration in Europe.

INTRODUCTION: Current external quality assurance schemes have been classified into six categories, according to their ability to verify the degree of standardization of the participating measurement procedures. SKML (Netherlands) is a Category 1 EQA scheme (commutable EQA materials with values assigned by reference methods), whereas SEQC (Spain) is a Category 5 scheme (replicate analyses of non-commutable materials with no values assigned by reference methods). AIM: The results obtained by a group of Spanish laboratories participating in a pilot study organized by SKML are examined, with the aim of pointing out the improvements over our current scheme that a Category 1 program could provide. METHOD: Imprecision and bias are calculated for each analyte and laboratory, and compared with quality specifications derived from biological variation. RESULTS: Of the 26 analytes studied, 9 had results comparable with those from reference methods, and 10 analytes did not have comparable results. The remaining 7 analytes measured did not have available reference method values, and in these cases, comparison with the peer group showed comparable results. The reasons for disagreement in the second group can be summarized as: use of non-standard methods (IFCC without exogenous pyridoxal phosphate for AST and ALT, Jaffé kinetic at low-normal creatinine concentrations and with eGFR); non-commutability of the reference material used to assign values to the routine calibrator (calcium, magnesium and sodium); use of reference materials without established commutability instead of reference methods for AST and GGT, and lack of a systematic effort by manufacturers to harmonize results. CONCLUSIONS: Results obtained in this work demonstrate the important role of external quality assurance programs using commutable materials with values assigned by reference methods to correctly monitor the standardization of laboratory tests with consequent minimization of risk to patients.

Is an intestinal permeability test a valid marker for slight dietary transgressions in adolescents with coeliac disease?

Adolescents with coeliac disease often fail to adhere to a strict gluten free diet. The effectiveness of intestinal permeability to sugars as a marker of slight dietary transgressions by such adolescents was assessed. Severe dietary transgressions were excluded from the study. Subjects were divided into two groups according to whether they committed slight dietary transgressions or adhered to a strict gluten free diet. A reference group of preadolescents with coeliac disease was also included in the study. Intestinal permeability and antigliadin antibody tests were performed on all patients. The diagnostic marker of intestinal permeability was excellent in the reference group. Neither the intestinal permeability test nor antigliadin antibody tests, however, succeeded in discriminating between the two groups of adolescents considered in this study. In conclusion the intestinal permeability test is not a valid marker for slight dietary transgression in such patients.

Alcoholic cognitive deterioration and nutritional deficiencies.

Chronic alcoholic patients frequently exhibit a mild to moderate cognitive impairment that has been related to Wernicke-Korsakoff encephalopathy and attributed tentatively to nutritional and vitamin deficiencies. To elucidate the possible relation between alcoholic cognitive deterioration (ACD) and nutritional and vitamin deficiencies, several tests of intelligence and memory were administered to 54 chronic alcoholic patients and 30 controls. Serum levels of thiamine, folic acid, vitamins B12, A, and E, and certain nutritional indexes were determined in most of the subjects. The alcoholics scored significantly lower in intellectual and visuospatial tasks but not in verbal memory tasks. They had a lower serum level for thiamine but not of the remaining vitamins. However, the correlations between serum thiamin and cognitive performance scores were low, and according to stepwise regression analysis, duration of alcohol intake and education were the variables with predictive value for intellectual and memory test performance. These results suggest that serum thiamin deficiency is not the main pathogenetic factor related to ACD.

Peripheral iron metabolism in patients with focal cerebral ischemia.

To ascertain the possible role of iron as a risk factor for cerebral ischemia, we studied the serum levels of iron, transferrin and ferritin in 42 patients between the third and seventh days after a cerebral ischemic event (transient ischemic attack, reversible ischemic neurological deficit or cerebral infarction) and in 62 matched controls. The serum levels of iron did not differ significantly between cerebral ischemic patient and control groups. Serum transferrin levels were lower and ferritin higher in stroke patients than in controls. These values were not influenced by age, blood pressure, or smoking and alcohol drinking habits. These results suggest that iron stores could be related to the risk for cerebral ischemia.

Decreased serum selenium concentrations in patients with Parkinson's disease.

Selenium is an essential component of the antioxidant enzyme glutathione peroxidase. The activity of this enzyme is reduced in the substantia nigra of patients with Parkinson's disease (PD), but the results of studies on erythrocytes are controversial. We compared the serum levels of selenium and the 24 h urinary selenium excretion (measured by hydride generation atomic absorption spectrophotometry) in 29 PD patients and 30 matched controls. Serum selenium levels were significantly lower in PD patients than in controls (34.6 ± 2.35 and 45.2 ± 3.83 µg/l, p < 0.05) while urinary excretion was similar for both groups (47.1 ± 6.25 and 45.5 ± 5.38 µg/24 h). These values were not influenced by antiparkinsonian drugs, and they did not correlate with age, age at onset and duration of the PD, scores of the Unified PD Rating Scale or the Hoehn and Yahr staging in the PD group. These results might suggest a possible role of low serum selenium levels in the risk for, or a consequence of the oxidative stress in PD.

Serum levels of nitrates in focal cerebral ischemia.

To ascertain the possible role of nitric oxide (NO) as risk factor for cerebral ischemia, we studied the serum levels of nitrate (oxidation product that provides an indirect estimation of NO), in 49 patients between the 3rd and 7th days after a cerebral ischemic event (TIAs, RIND and cerebral infarction) and in 65 matched controls. The serum levels of nitrate did not differ significantly between cerebral ischemic patient and control groups (42.6 ± 334 and 44.8 ± 2.67 ± µmol/l, respectively). They were not influenced by age, blood pressure, or smoking and alcohol-drinking habits. These results suggest that serum levels of nitrate are apparently unrelated to the risk for cerebral ischemia.

[Evaluation of a totally automated alternative system for determining the rate of erythrocyte sedimentation]. / Valoración de un sistema alternativo totalmente automatizado para la determinación de la velocidad de sedimentación globular.

The Ves-MaticR system (VS) for evaluating erythrocyte sedimentation rate (ESR) is compared to Westergren method. A good correlation between both methods is obtained in spite of small differences observed; the accuracy of the VS is also studied. Concerning the time variation, a small reduction of the ESR in the first seven hours is observed, being significant at 24 hours. The most important advantages of the VS are the near complete disappearance of the contamination risk, the reduction of the time employed in setting up, sedimentation and reading, and the reliability.