Clin Chem Lab Med Research Articles

Lack of association between DAZ gene methylation patterns and spermatogenic failure

Abnormal DNA methylation of the male germ line is proposed as a possible mechanism causing compromised spermatogenesis in some men diagnosed with idiopathic infertility. Previous studies suggested that aberrant DNA methylation of several genes is associated with disruptions in spermatogenesis. However, little information is available on DNA methylation patterns of testis-specific genes in idiopathic male infertility. To investigate the association between DAZ gene methylation patterns and spermatogenic failure, we performed an analysis of methylation patterns in 174 idiopathic infertile patients and 58 fertile controls using bisulfite-modified sequencing. We found that the methylation patterns of CpG island (CGI) in the DAZ gene promoter region were different between somatic cells and spermatic cells in the control group. DAZ gene methylation patterns among groups with different spermatogenic status were the same in somatic cells, completely methylated, and in spermatic cells. The results were concordant, except for the group with azoospermia (AZ) which were completely unmethylated. Our data indicate that the methylation patterns of the DAZ gene are not associated with spermatogenic failure. This suggests that epigenetic modification of DAZ is unlikely to be involved in the etiology of spermatogenic failure.

Evaluation of five routine glucose methods on an Olympus AU5400 analyzer using the CDC hexokinase reference method

In China, two common methods are used to detect serum glucose, glucose oxidase (GOD) and hexokinase (HK) methods. We evaluated five commercially available glucose reagent kits with the Center of Disease Control (CDC) HK reference method. Five routine methods were compared with the HK reference method according to Clinical and Laboratory Standards Institute (CLSI) guideline (EP9-A2). These five routine methods were performed on an "open channel" Olympus 5400 analyzer. All five evaluated methods were closely correlated to the reference method (r > 0.9998). Compared to the HK reference method, the Olympus-HK method and Roche-GOD method showed a very small positive bias (mean +/- SD: 0.08 +/- 0.09 mmol/L and 0.03 +/- 0.08 mmol/L); while the Biosino-HK method, Osborne-GOD method and Baiding-GOD method showed a very small negative bias (mean +/- SD: -0.07 +/- 0.06 mmol/L, -0.09 +/- 0.07 mmol/L and -0.01 +/- 0.04 mmol/L). All five reagents we evaluated using matching calibrators showed good correlation to the reference method and small bias. These five glucose reagents are acceptable for use in clinical laboratories.

Simultaneous determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography with fluorescence detection

To provide a more comprehensive clinic marker of tryptophan (TRP) catabolism in patients with systemic lupus erythematosus (SLE), we developed a simple and efficient method that simultaneously measured serum TRP, kynurenine (KYN), and kynurenic acid (KYNA) using high performance liquid chromatography with fluorescence detection (HPLC-FD). A simple and specific high performance liquid chromatography (HPLC) method was developed for simultaneously quantitative determination of TRP, KYN and KYNA with fluorescence detection (FD) using programmed wavelength and on-column fluorescence derivatization. Thirty patients with SLE and 80 healthy control subjects were analyzed for serum TRP metabolites using the assay we developed. The tryptophan breakdown index (TBI) and neuroprotective ratio (NPR) were calculated. The retention time of KYN, KYNA and TRP were 8.5 min, 13.7 min and 17.6 min, respectively. The linear range for TRP was 0.245-196 micromol/L, the limit of detection (LOD) was 0.001 micromol/L and average recovery was 103.71%. The linear range for KYN was 0.049-98 v/L, the LOD was 0.0245 micromol/L, and average recovery was 97.45%. The linear range for KYNA was 1.05-2093 nmol/L, the LOD was 0.05 nmol/L, and average recovery was 100.60%. Inter-day and intra-day relative standard deviations (SDs) were <5%. Phenylalanine, tyrosine, 5-hydroxytryptamine and creatinine did not interfere with the method. The results showed great differences in TRP, KYN and KYNA contents and TBI between patients with SLE and healthy controls, but little difference in NPR. The method is simple, fast, accurate, and meets the requirements for simultaneous determination of TRP, KYN and KYNA in serum.

Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids

We evaluated the body fluid (BF) mode on the new Sysmex XE-5000 analyzer. Red (RBC) and white blood cell (WBC) (differential) counts of BFs (139 patient samples and 87 normal samples) were measured and compared to the Fuchs-Rosenthal chamber and stained cytospin slides. Extended cell counting using the BF mode was noted to have an improved WBC detection limit (CV(20)%) of 10 x 10(6)/L. Excellent agreement with the manual method was observed for most BFs [mean bias +2 to 6 x 10(6)/L for cerebrospinal fluid (CSF) and -1 to 12 x 10(6)/L for other fluids]. In CSF, the BF-mode counted more WBC (polymorphic nuclear cells) compared with the manual method (mean bias +5 to 6 x 10(6)/L), especially in samples with low cell counts (<20 x 10(6)/L). Carry over was negligible (mostly <0.17%) and linearity was excellent (mean bias <5%). The reference ranges for CSF (n=87) were RBC 0 x 10(6)/L, WBC and mononuclear <7 x 10(6)/L, and polymorph nucleated cells <3 x 10(6)/L. The BF mode on the Sysmex XE-5000 offers rapid and accurate RBC and WBC (differential) counts in clinically relevant concentration ranges in CSF and other fluids. In addition, the exclusion of high fluorescent cells, such as mesothelial cells and macrophages from WBC counting may reduce the number of manual analyses in pleural fluids and ascites.

Reference intervals for brain natriuretic peptide in healthy newborns and infants measured with an automated immunoassay platform

In order to assess the reference intervals for B-type natriuretic hormone (BNP) in the first days of life, we measured peptide concentrations using the fully automated Access platform. Plasma BNP was measured in 188 apparently healthy newborns and infants throughout the first month of extra-uterine life, as well as in 245 healthy infants ranging from 1 month to 12 years of age. BNP showed the highest concentrations in the first 2 days of life, with a progressive decline afterwards. Moreover, BNP values in the first week of life were significantly higher (p<0.0001) than values observed in the next periods. As a result, a significant negative correlation was found between BNP and age values when considering all 433 samples (rho=-0.816, p<0.0001 by the Spearman rank correlation test). There was no significant difference between BNP values found in males and females. According to this data, our study indicates that at least two reference intervals should be used for newborns and infants. The first, with higher BNP values for neonates in the first week of extra-uterine life, and the other, with lower BNP values for infants aged 2 weeks to 12 years.

State of the art in therapeutic drug monitoring

Therapeutic drug monitoring (TDM) is a multidisciplinary activity. Because laboratory reports are part of the patient's chart, some clinical information is required. In order to guarantee quality and safety, an increasing number of TDM departments have implemented a quality management system. The aim of the present article is to review the three phases of TDM: the pre-analytical, analytical and post-analytical phases. In the pre-analytical phase, it is necessary to acquire a valid specimen collected at the specific time window. Analytical methods should be validated, assessing possible interfering substances. The objective of the post-analytical phase is the final report, which should include correct interpretation, as well as possible advice. Appropriate pharmacokinetic interpretation avoids unnecessary costs and leads to clinical benefits.

The underestimated problem of using serum magnesium measurements to exclude magnesium deficiency in adults; a health warning is needed for “normal” results

A major use of serum magnesium measurements in clinical practice is to identify patients with deficiency. However, numerous studies have shown that magnesium deficiency is common and may be present in over 10% of hospitalized patients, as well as in the general population. An important cause for under diagnosis of deficiency is that serum magnesium, the most commonly used test, can be normal despite negative body stores. This article focuses on the limitations of "normal" magnesium results and highlights the importance of lifestyle or "modus vivendi" as a pragmatic means of identifying those individuals potentially at risk for negative body magnesium stores. Researched peer reviewed articles on magnesium published between 1990 and 2008 in MEDLINE and EMBASE, using database keywords "magnesium, deficiency, diagnosis, treatment and hypomagnesaemia". Bibliographies of retrieved articles have been searched and followed. We have also performed a manual search of each individual issue in which most of these reports have appeared. In 183 peer reviewed studies published from 1990 to 2008, magnesium deficiency was associated with increased prevalence and risk in 11 major conditions. Similarly, in 68 studies performed over the same period, magnesium deficiency was found to predict adverse events and a decreased risk of pathology was noted when supplementation or treatment was instituted. The perception that "normal" serum magnesium excludes deficiency is common among clinicians. This perception is probably enforced by the common laboratory practice of highlighting only abnormal results. A health warning is therefore warranted regarding potential misuse of "normal" serum magnesium because restoration of magnesium stores in deficient patients is simple, tolerable, inexpensive and can be clinically beneficial.

Evaluation of C-reactive protein and serum amyloid A in the detection of inflammatory and infectious diseases in children

Various biomarkers have been proposed to help diagnose inflammatory and infectious diseases. The goal of this study was to evaluate the effectiveness of some of these markers in children seen in an emergency service, and to compare a number of routinely used tests with measurements of serum amyloid A (SAA) and C-reactive protein (CRP) levels. A total of 83 children with clinically suspected inflammation (n=17) or infection (n=66) who had been seen at the emergency department were evaluated. Complete blood count, erythrocyte sedimentation rate (ESR), CRP (using two methods) and SAA measurements were performed. There was no difference in the parameters studied between the inflammation and infectious groups. However, when the infectious group was subdivided into bacterial (n=21) and viral infections (n=43), significantly higher leukocyte, CRP and SAA values were observed in the former group. CRP was the most accurate indicator of inflammatory and infectious processes [Kappa concordance index (KCI)=0.89], followed by SAA (KCI=0.73) and ESR (KCI=0.73). The worst results were obtained for the immature/total neutrophil ratio (KCI=0.02). Measurement of CRP showed the best performance for indicating infectious or inflammatory processes. ESR and SAA had good concordance and yielded very similar results. CRP may be included in protocols for pediatric assessment in emergency departments.

FOXL2 mutations in Taiwanese patients with blepharophimosis, ptosis, epicanthus inversus syndrome

Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) is an autosomal dominant developmental disorder that includes an eyelid malformation associated with (type I) or without (type II) premature ovarian failure (POF). Mutations in the forkhead transcription factor 2 (FOXL2) gene, a member of winged/forkhead transcription factor family, are responsible for both types of BPES. The purpose of this study was to identify mutations in FOXL2 in Taiwanese patients with BPES. The karyotype and genomic DNA was prepared from the leukocytes of peripheral venous blood samples. The coding and flanking region sequences of FOXL2 were analyzed by directed or cloning sequencing. The karyotypes of these patients did not show significant variation, especially on the 3q23 region. Two mutations in FOXL2 were identified in two familial cases. One was c.855-871dup (17-bp insertion) associated with POF. The other was c.384G>A (TGG>TGA), a novel mutation that resulted in non-sense changes of the encoded protein, i.e., p.W128X. Our results expand the spectrum of FOXL2 mutations and confirm the mutation hotspot in FOXL2 in Taiwanese BPES patients.

Association of matrix metalloproteinase 9 genotypes and cardiovascular disease risk factors with serum matrix metalloproteinase 9 concentrations in Taiwanese individuals

Circulating concentrations of matrix metalloproteinase 9 (MMP-9) are associated with cardiovascular disease mortality in patients with coronary artery disease. We investigated the determinants of MMP-9 concentrations by analyzing MMP-9 genotypes and risk factors for cardiovascular disease. A total of 596 individuals were recruited for this study. Six single nucleotide polymorphisms (SNP) with coverage of the MMP-9 gene region were analyzed; and two genotypes, rs3918242-TT and rs2274756-AA, which were in nearly complete linkage disequilibrium, were associated with higher MMP-9 values (p=0.007 and p=0.008, respectively). In age- and gender-adjusted regression models, MMP-9 concentrations were positively associated with the homeostasis model assessment of insulin resistance (HOMA-IR) index and triglyceride concentrations, fasting serum insulin, fasting plasma glucose, C-reactive protein, fibrinogen, and serum amyloid A. By multivariate analysis, rs2274756 genotypes, age, smoking status, fibrinogen and fasting plasma glucose concentrations were all independently associated with MMP-9 (p=0.007, p=0.033, p=0.003, p=0.013, and p=0.012, respectively). The rs2274756-AA and rs3918242-TT genotypes, younger age, current smoking status and increased fasting plasma glucose, and fibrinogen concentrations were independently associated with high serum MMP-9 concentrations in Taiwanese individuals.

C-reactive protein variants are not associated with susceptibility to stroke and stroke recurrence

C-reactive protein (CRP) has been shown to contribute to the risk of cardiovascular disease. Several single nucleotide polymorphisms (SNPs) at the CRP locus have been found to be associated with CRP concentrations. We hypothesized that genetic variants associated with high CRP may confer a greater risk of stroke and recurrence. The hypothesis was tested in a case-control study and during follow-up of the case population. Two genetic variants associated with serum CRP in the CRP gene were genotyped in a Chinese case-control study comprised of 1572 stroke patients and 1485 controls. The case population was followed for a median of 4.5 years (range, 0.1-6.0 years). During a median 4.5-year follow-up for 1526 stroke patients, 299 recurrent strokes were identified. No association was found between the SNPs or haplotypes of the CRP gene and stroke and its recurrence. Although these variants and corresponding haplotypes in the CRP gene are associated with serum CRP concentrations, our study does not support that variants and corresponding haplotypes studied here have a major influence on risk of stroke and stroke recurrence. Therefore, we speculate that CRP is not a causal factor for stroke.

Immunoglobulin M antibodies to aquaporin-4 in neuromyelitis optica and related disorders

Neuromyelitis optica (NMO, Devic syndrome) is an inflammatory disorder of the central nervous system of putative autoimmune etiology that primarily affects the optic nerves and spinal cord. NMO is frequently associated with immunoglobulin G (IgG) antibodies to aquaporin-4 (AQP4-IgG), which are thought to be involved in the patho-genesis of the disease. The frequency and diagnostic relevance of immunoglobulin M (IgM) antibodies to aquaporin-4 (AQP4-IgM) in patients with NMO is essentially not known. Testing for AQP4-IgM may be of importance since 20%-30% of patients with NMO are negative for AQP4-IgG. Moreover, IgM antibodies are more potent activators of complement compared with IgG, and are detectable at NMO lesional sites. Serum samples from 42 patients with NMO spectrum disorders (NMOSD) and from 66 controls were tested for IgM AQP4-Ab using a cell-based assay employing HEK293 cells transfected with human full length AQP4. To control for possible interactions between IgG and IgM, serum was depleted of IgG prior to testing by indirect immunofluorescence. IgM AQP4-Ab were detectable in 4/42 samples from patients with NMOSD, but in none of the 66 control samples. In three patients, titers were higher following depletion of total IgG from the samples. One sample was positive only after precipitation of total IgG. AQP4 antibodies of the IgM class exist in almost 10% of patients with NMO and might contribute to lesion pathology. Routine testing for AQP4-IgM appears to not be justified, as all AQP4-IgM positive patients were also positive for AQP4-IgG, and none of the AQP4-IgG negative samples were positive for AQP4-IgM.

Combined analysis of survivin autoantibody and carcinoembryonic antigen biomarkers for improved detection of colorectal cancer

Survivin is a member of the family of inhibitor of apoptosis proteins that is overexpressed in several human tumors. Previous studies have found that overexpression of survivin in cancer cells induces an antibody response. We compared 232 serum samples from colorectal cancer (CRC) patients and 365 samples from healthy volunteers using an in vitro enzyme-linked immunosorbent assay to evaluate the survivin autoantibody response in patients. The sensitivity of the anti-survivin response from patients with CRC was 56.9%, and the specificity was 64.1%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in these same serum samples, the values for sensitivity and specificity were 40.9% and 86.6%, respectively. Combined detection using survivin autoantibodies and CEA produced better sensitivity (51.3%) and specificity (89.9%) compared to the sensitivity of CEA (40.9%) and the specificities of the individual markers (64.1% and 86.6%, respectively). The area under a receiver operating characteristic curve was 0.617 for survivin autoantibodies, 0.630 for CEA and 0.694 for both markers together. A positive association between autoantibodies against survivin and preoperative CEA concentrations in sera of patients with CRCs was established. Our results suggest that analysis of both parameters would assist in screening patients with CRC.

Factors influencing serum total tryptase concentrations in a general adult population

Serum tryptase measurements are increasingly being used in clinical practice. However, there is a paucity of studies focused on factors that influence tryptase concentrations. We investigated the relationships between total tryptase concentrations and age, gender, atopy, alcohol consumption, smoking, and common metabolic abnormalities in adults. This was a cross-sectional study of a random sample from an adult population (n=420, age 18-92 years, 45% males) from a single municipality. Serum total tryptase was measured using a fluoro-enzyme immunoassay. The median tryptase concentration was 5.1 microg/L (range, <1-30.7 microg/L). There was a significant (p<0.0001) continual increase in tryptase with age; the median concentrations were 4.0 microg/L in 18-30 years old and 6.6 microg/L in those >80 years. Female gender and heavy ethanol use were negatively and independently associated with tryptase concentrations. Tryptase was higher in individuals that were non-atopic, overweight, or had metabolic syndrome compared to individuals that were atopic, normal weight, or did not have the metabolic syndrome. However, these associations were attenuated after adjusting for age. Serum total tryptase concentrations in adults are not significantly modified by atopy status, but may be modified by lifestyle factors, such as alcohol consumption and gender. Serum total tryptase concentrations are particularly dependent on age.

Cardiovascular risk factors and γ-glutamyltransferase fractions in healthy individuals

Serum gamma-glutamyltransferase activity (GGT), even when within its normal reference range, catalyzes low density lipoprotein oxidation in vitro and predicts cardiovascular events. Of the four GGT fractions (b-GGT, m-GGT, s-GGT, and f-GGT) recently identified in blood, only b-GGT is found within atherosclerotic plaques. Our goal was to identify the determinants of the GGT fractions (b-, m-, s-, and f-GGT) and their association with established cardiovascular risk factors in healthy subjects. Multiple linear regression analysis was applied to estimate the association of fractional GGT with gender, age, body mass index, arterial pressure (AP), plasma glucose, alanine aminotransferase (ALT), high and low density lipoproteins (LDL-C) cholesterol (HDL-C), triglycerides (TG) and C-reactive protein (CRP) in 200 healthy subjects. All GGT fractions were associated with ALT; b-GGT with AP, TG, and CRP; m-GGT with LDL-C, TG and CRP; s-GGT with TG and CRP, and f-GGT only with LDL-C, whereas gender was associated with s-GGT and f-GGT only. In healthy individuals, cardiovascular risk factors are associated with high molecular weight GGT fractions, namely with b-GGT, the only form present within the plaque. This finding adds to the present knowledge concerning the relevance of GGT, within its reference range, for atherosclerosis-related events.

A new statistical method for evaluating long-term analytical performance of laboratories applied to an external quality assessment scheme for flow cytometry

The Belgian External Quality Assessment Scheme for Flow Cytometry evaluates the long-term analytical performance of participating laboratories by calculating a regression line between the target and reported values of each parameter for each laboratory during the past 3 years. This study aims to develop a method to find laboratories with aberrant variability or bias using robust techniques and to obtain robust estimates of the variability. A method is proposed to find outliers with respect to the individual regression line, followed by a step to find regression lines with excessive variability and finally a step to find regression lines with high bias. The model was applied to the results obtained by 52 laboratories for CD4%. From the 1340 data points, 35 were determined to be regression outliers. The second step revealed one regression line with excessive variability; the third step detected three regression lines with exceeding bias. The methodology allows assessment of the long-term performance of laboratories, taking into account samples with different target values. Outliers in the first step indicate accidental mistakes, outliers in the second and third step point to high analytical variability or bias.

Extracellular haemoglobin, oxidative stress and quality of red blood cells relative to perioperative blood salvage

The quality of blood-pack units in perioperative blood salvage was studied with respect to the presence of extracellular haemoglobin (Hb) and in terms of oxidative stress and the mechanical properties of red blood cells (RBC). The study was performed on blood-pack units and patients' blood after retransfusion. Results were compared to those obtained in patients prior to autotransfusion. Free Hb, non-protein thiols (as antioxidant marker) and markers of oxidative stress (conjugated dienes, thiobarbituric reactive substances and protein carbonyl content) were measured. The mechanical properties of RBC were evaluated by their osmotic fragility, membrane fluidity by measuring the fluorescence anisotropy of an extrinsic probe and permeability by measurement of extracellular K(+) and lactate dehydrogenase. Despite washing, extracellular Hb concentrations remained high (up to 0.7 g/L in blood-pack units) and was associated with a decrease of haptoglobin in patients, despite a concomitant inflammatory syndrome. In blood-pack units, we observed a decrease in antioxidant markers along with an increase in oxidative stress markers in association with an alteration of RBC membrane properties. Haemolysis must be limited during perioperative blood salvage in order to prevent exposure to oxidative stress and improve the efficiency of autotransfusion.

Association of hyperhomocysteinemia with left ventricular dilatation and mass in human heart

Hyperhomocysteinemia is a risk factor for ischemic heart disease. Several other mechanisms apply also to dilative types of heart failure of various, non-ischemic etiologies. We hypothesized that hyperhomocysteinemia is associated with left ventricular (LV) dilatation and hypertrophy in dilative cardiomyopathy. Homocysteine was measured in 66 individuals with suspected cardiomyopathy. Cardiac magnetic resonance imaging was used to assess LV volume, mass, and wall stress. Hyperhomocysteinemia (> 12 micromol/L) was found in 45 patients (68%). LV mass was greater in these patients compared with individuals with normal homocysteine (83+/-27 vs. 67+/-19 g/m(2); p<0.02). Homocysteine was increased in patients with increased brain natriuretic peptide > or = 100 pg/mL (18.3+/-5.9 vs. 14.9+/-5.1 micromol/L; p=0.018). LV mass, LV end-diastolic and end-systolic volume (LVEDV, LVESV) were significantly increased in individuals in the upper quartile compared with the lower quartile (90+/-25 vs. 65+/-18 g/m(2), p=0.021; 114+/-50 vs. 71+/-23 mL/m(2), p=0.042; 76+/-51 vs. 36+/-22 mL/m(2), p=0.045). LV dilatation (LVEDV > or = 90 mL/m(2)) was more common in hyperhomocysteinemia (> 12 micromol/L, p=0.0166). Normalized LV mass was correlated with homocysteine (r=0.346, p=0.065). Homocysteine was not significantly correlated with LVEDV (r=0.229, p=0.065), LV end-diastolic wall stress (r=0.226, p=0.069) and LV ejection fraction. Hyperhomocysteinemia appears to be, at least in part, involved in a disproportional LV dilatation, where the ensuing hypertrophy is not sufficient to compensate for the increased wall stress. A potential mechanism is the hyperhomocysteinemia associated increase in oxidative stress that favors muscle fiber slippage.

Comparison of different depletion strategies for improving resolution of the human urine proteome

Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated. To remove the most abundant proteins from a human urine sample, GenWay Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies. Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343+/-20.0 microg, 5.8%; 186.3+/-13.3 microg, 7.2%; 292+/-20.6 microg, 8.8% [mean+/- standard deviation (SD), CV%], for GenWay, Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468+/-21, 331+/-7, 368+/-22 and 304+/-7 (mean+/-SD) for GenWay, Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p<0.001 compared to the GenWay procedure. The sequential procedure of urine samples using multi-protein immuno-affinity depletion represents a valid tool for simplifying 2-DE analysis of the urine proteome. Particularly, the GenWay kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome.

Correlation between glucose and bupivacaine levels in cerebrospinal fluid after spinal anesthesia: glycorrhachia as predictor for duration of sensory block

Prediction of the duration of motor block after injection of a local anesthetic into cerebrospinal fluid (CSF) would be a very useful tool in clinical practice. However, previous attempts have not shown conclusive results. In this work, glycorrhachia is demonstrated to be an adequate predictive parameter after spinal anesthesia using 0.5% hyperbaric bupivacaine. Two mL of local anesthetic through a continuous spinal catheter was administered to 40 patients. CSF was sampled at different time intervals from the onset of infusion to motor recovery. CSF bupivacaine concentrations were measured using chromatography. An automated analyzer was used for determining glycorrhachia in the same samples. For all patients, good correlation (r(2)>0.95, p<0.05) was obtained. From these results, it was possible to develop a general model which establishes the relationship between CSF glucose and bupivacaine concentrations (R(2)=0.987, p<0.05). Motor block is reached when CSF glucose concentration is about 245 mg/dL (13.5 mmol/L), which corresponds to 35 microg/mL of bupivacaine. Glycorrhachia measured during surgical intervention in patients undergoing spinal anesthesia with hyperbaric bupivacaine provides a mechanism for predicting the duration of motor block in a rapid and simple manner.