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A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography-mass spectrometry (GC/MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules is available for separation and quantification by GC/MS. The urease method is a useful adjunct to newborn screening follow-up, and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. This method describes the urease treatment, derivatization, and the organic acids and other biochemical metabolites that can be identified.

BACKGROUNDCystic fibrosis (CF) is a complex autosomal recessive disease that continues to present unique diagnostic challenges. Because CF was first described in 1938, there has been a substantial growth of genetic and phenotypic information about the disorder. During the past few years, as more evidence has become available, a consortium of international experts determined that the 2008 guidelines from the CF Foundation needed to be reviewed and updated.CONTENTThe goal of this review is to highlight the latest advances in CF multidisciplinary care, together with the recent updates to the 2017 CF Foundation diagnostic guidelines.SUMMARYData from newborn screening programs, patient registries, clinical databases, and functional research have led to a better understanding of the CF transmembrane conductance regulator (CFTR) gene. Recent consensus guidelines have provided recommendations for clinicians and laboratorians to better assist with interpretation of disease status and related CF mutations. The highly recommended Clinical and Functional Translation of CFTR project should be the first resource in the evaluation of disease severity for CF mutations. Screen-positive newborns and patients with high clinical suspicion for CF are always recommended to undergo confirmatory sweat chloride testing with interpretations based on updated reference intervals. Every patient diagnosed with CF should receive genotyping, as novel molecular therapies are becoming standard of practice. The future of CF management must consider healthcare system disparities as CF transitions from a historically childhood disease to a predominantly adult epidemic.

Context.—Discrepant results for serum constituents were observed among peer groups in the College of American Pathologists Comprehensive Chemistry Survey.Objectives.—To assess the performance of serum albumin and total protein measurement procedures and to evaluate the commutability of the conventional survey specimens.Design.—A fresh frozen, off-the-clot serum sample was included along with 4 conventional survey specimens. The fresh frozen, off-the-clot serum sample was prepared in a manner expected to confer commutability with native clinical samples.Results.—For the fresh frozen, off-the-clot serum sample, the mean values for 17 peer-groups were −0.07 to 0.32 g/dL from the bromocresol green albumin designated comparison method, whereas 4 VITROS (Ortho Clinical Diagnostics, Rochester, New York) peer groups differed by −0.29 to −0.37 g/dL (15 of 21 differences [71%] had P < .001). For bromocresol purple albumin methods, the mean differences from the designated comparison method from 8 peer groups were 0.25 to 0.47 g/dL (all had P < .001). For total protein methods, 23 peer group mean values were −0.07 to 0.15 g/dL from the reference measurement procedure (12 of 24 [50%] had P < .001). The Beckman (Fullerton, California) Synchron LX20 had a bias of −0.30 g/dL (P Conclusions.—One (2.2%) of the instrument systems (Synchron) using bromocresol green and none (0%) of the instrument systems using bromocresol purple had satisfactory total-error performance for albumin measurement. Differences in results between bromocresol green and bromocresol purple methods precluded using common reference intervals for interpreting results for serum albumin. Eight of 9 instrument systems (86.5%) had satisfactory total-error performance for total protein measurement.

BACKGROUND Reference intervals from children are limited by access to healthy children and their limited blood volumes. In this study we set out to fill gaps in pediatric reference intervals for amino acids and steroid hormones using dried blood spots (DBS) from a cohort of the National Children's Study. METHODS Deidentified DBS annotated with age, birthweight, sex, and geographic location were obtained from 310 newborns aged 0–4 days and analyzed for 25 amino acids and 4 steroid hormones using LC-MS/MS. Nonparametric statistical approaches were used to generate the 2.5th–97.5th percentile distributions for newborns. Paired plasma/DBS specimens were used to mathematically transform DBS reference intervals to corresponding plasma intervals. RESULTS 10 of 25 DBS amino acid distributions were dependent on sex. There was little correlation with age, birthweight, or geographic location over the first 4 days of life. In most cases, transformation of DBS distributions to plasma distributions faithfully reflected independent studies of newborn plasma amino acid distributions. In general newborn steroid distributions were negatively correlated with age and birthweight over the first 4 days of life. Data distributions for the 4 steroids were not found related to geographic location, but testosterone concentrations displayed sex dependence. Transformation of DBS distributions to plasma intervals did not faithfully replicate other neonate steroid reference intervals determined directly with plasma. CONCLUSIONS These data demonstrate the feasibility and utility of deriving newborn reference intervals from large numbers of archived DBS samples such as those obtained from the National Children's Study biobank.

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions.Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.

In 2003, the Chemistry Resource Committee of the College of American Pathologists introduced a new specimen in the Neonatal Bilirubin Survey. The specimen, consisting of human serum enriched with unconjugated bilirubin and thus resembling a clinical specimen, brought about an improvement in the accuracy of the measurement of bilirubin by laboratories participating in the Neonatal Bilirubin Survey. There was also an improvement in the specificity of methods measuring direct bilirubin. However, persisting inaccuracies and variability in laboratory performance have been traced to calibrators consisting of bovine serum spiked with unconjugated bilirubin and ditaurobilirubin; bovine serum causes underestimation of both bilirubins by 8 major chemical analyzers. To eradicate inaccuracy calibrators and Survey specimens should be made in human instead of bovine serum.

To determine the relationship between BiliChek TcB (Respironics, Marietta GA) and Doumas reference serum or plasma total bilirubin (TSB).Pooled samples with values assigned by the Doumas reference method were used to establish the relationship between a local laboratory and reference Doumas TSB. We then established the relationship between TcB and TSB in the 3 months before and after reassignment of calibrator setpoints undertaken to match the local laboratory to Doumas reference bilirubin values.Before calibrator setpoint reassignment TSB as measured in our laboratory overestimated Doumas reference bilirubin. After calibrator adjustment laboratory TSB was within 1.7-6.8 micromol/L (0.1-0.4 mg/dL) of Doumas reference values. Mean bias between BiliChek TcB and TSB was 42.8+/-22.2 micromol/L (2.5+/-1.3mg/dL) (n=94) before and 49.6+/-22.2 micromol/L (2.9+/-1.3mg/dL) (n=115) after calibration adjustment.BiliChek TcB significantly overestimates TSB as measured by the Doumas reference method.

Context.—In 2003 the Chemistry Resource Committee of the College of American Pathologists introduced a commutable specimen in the Neonatal Bilirubin Surveys. This specimen was intended to help evaluate all bilirubin methods.Objective.—To evaluate the effect of commutable specimens on the performance of selected clinical analyzers in measuring neonatal bilirubin from 2003 through 2006.Design.—A human serum–based specimen enriched with unconjugated bilirubin in human serum has been included since 2003 in the Neonatal Bilirubin Surveys. The bilirubin values of these specimens were determined by the reference method and used to evaluate results reported by various chemistry analyzers.Results.—Coefficients of variation for College of American Pathologists All Data ranged from 4.9% to 6.2% for the Neonatal Bilirubin Survey. However, coefficients of variation for the 4 major instrument groups (Dimension, Olympus, Synchron, and Vitros), which report 65% of all results, varied from 2% to 3%. College of American Pathologists All Data mean bilirubin values were within 0.46 mg/dL (7.8 μmol/L) of the reference method mean in 2003; in subsequent years these differences became larger, peaking at 1.87 mg/dL (32 μmol/L) in 2005.Conclusions.—The large systematic error of bilirubin measurements is due primarily to failure of instrument manufacturers to produce reliable bilirubin calibrators. Primary calibrators should consist of human serum enriched with unconjugated bilirubin. Bilirubin values must be assigned by the reference method, the performance and robustness of which are reported in this article. Secondary calibrators distributed to users must be traceable to primary calibrators.

Background Lactate is commonly used in septic patients and is a viable biomarker for trauma patients. Its pre-hospital use could assist triaging and managing patients with these conditions. Methods We evaluated the analytical performance of the point-of-care (POC) StatStrip Xpress Lactate Meter (Nova Biomedical) and compared it to the ABL 800 (Radiometer). We measured lactate in 250 adult and 250 pediatric whole blood samples in 2 laboratories. The performance of the POC meter was assessed by traditional linear regression and Bland-Altman plots, and locally-smoothed (LS) median absolute difference and maximum absolute difference (MAD and MaxAD) curves. Results The StatStrip was linear with acceptable reproducibility at clinically relevant concentrations. Correlation with the ABL800 showed a negative bias for both populations with slope, bias ± SD (% bias) of 0.78, − 0.4 ± 0.7 (− 14.5%) in children and 0.80–0.3 ± 0.6 (− 13.3%) in adults. The proportional bias appeared more significant at concentrations > 4 mmol/l (36.0 mg/dl). The StatStrip misclassified 7.6 and 8.8% pediatric and adult samples, respectively, to lower risk categories defined using guidelines driven cut-offs. The LS MAD curves identified one breakout, concentration where the LS MAD exceeds the total allowable error limit of 0.3 mmol/l (2.7 mg/dl), at lactate concentrations of 3.8 and 3.2 mmol/l (34.2 and 28.8 mg/dl) in the pediatric and adult curves, respectively. Breakthroughs, points at which the LS MaxAD curve exceeds the 95th percentile of MaxADs, occur at concentrations above 7.5 mmol/l (67.6 mg/dl) for both populations where the performance of the POC meter became erratic. We concluded that if serial lactate measurements are performed, the same method should be used for baseline and follow up measurements. The LS MAD and LS MaxAD curves allowed visual and quantitative mapping of the performance of the lactate POC meter over the range of concentrations measured. Conclusions This approach seems useful for the identification of points at which the performance of a POC meter differs significantly from a comparison method and thresholds of poor analytical performance.

Background: The analytic performance and accuracy of drug detection below Substance Abuse and Mental Health Services Administration (SAMHSA) cutoffs is not well known. In some patient populations, clinically significant concentrations of abused drugs in urine may not be detected when current SAMHSA cutoffs are used. Our objectives were to define the precision profiles of three immunoassay systems for drugs of abuse and to evaluate the accuracy of testing at concentrations at which the CV was Methods: Drug-free urine was supplemented with analytes to assess the precision in three commercial drugs-of-abuse immunoassay systems below the SAMHSA-dictated cutoffs for amphetamines, opiates, benzoylecgonine, phencyclidine, and cannabinoids. Consecutive urine samples with signals associated with a CV Results: The CV of all immunoassay systems tested remained Conclusion: The precision of three commercial immunoassay systems for drugs-of-abuse screening is adequate to detect drugs below SAMHSA cutoffs. Knowledge of the positive predictive values of screening immunoassays at lower cutoff concentrations could enable efficient use of confirmatory testing resources and improved detection of illicit drug use.

Objectives To assess the performance of the Doumas bilirubin reference method. Design and methods Ring trails using pooled patient specimens, a calibrator and human sera enriched with unconjugated bilirubin were analyzed in five laboratories using the Doumas bilirubin reference method. Results The coefficient of variation for the linear measurement range between laboratories ranged from 1–3%. Conclusions The Doumas bilirubin reference method is robust and reproducible. Bilirubin results using this method may be used in the development of more accurate and reliable calibrators.

The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V.P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.

In 2003, the College of American Pathologists (CAP)1 included in the Neonatal Bilirubin Surveys a human serum–based sample enriched solely with unconjugated bilirubin (UBIL). This sample, shipped frozen in a cold pack, resembled closely a clinical sample from a healthy neonate and was commutable, as expected. The mean of all results submitted to CAP (CAP All Data) for bilirubin (199.4 mg/L) was close to that of the reference method (194.4 mg/L)(1). In subsequent years, the bias has increased(2). Most recently (2009, neonatal bilirubin NB-01), the difference between the mean value for CAP All Data and the reference method value was 9 mg/L, and the differences for the Olympus, Ortho Vitros, and Siemens ADVIA assays were closer to 25 mg/L. Because ditaurobilirubin (DTB), which is present in most commercial calibrators, and bovine serum, which is used instead of human serum for preparing calibrators, have been shown to interfere with the measurement of bilirubin(3), we investigated the effects of DTB and bovine serum on the measurement of total bilirubin by diazo methods. UBIL and DTB were purchased from Lee Biosolutions. Human serum pools (nos. 1 and 2) were obtained from volunteers from the laboratory personnel of the Children’s Hospital of Wisconsin. Bovine sera and donor calf serum were purchased from Sigma–Aldrich and MP Biomedicals. Fresh bovine serum was obtained from a local abattoir in Milwaukee, Wisconsin. Stock solutions of UBIL and DTB were prepared in all of the above-described protein matrices. The concentrations of UBIL (210 mg/L) …

A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography-mass spectrometry (GC-MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds, which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules are available for separation and quantitation by GC-MS. The urease method is a useful adjunct to newborn screening follow-up and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. The method below describes the urease treatment, derivatization, and the organic acids, and other biochemical metabolites that can be identified.

A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography-mass spectrometry (GC-MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds, which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules are available for separation and quantitation by GC-MS. The urease method is a useful adjunct to newborn screening follow-up and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. The method below describes the urease treatment, derivatization, and the organic acids, and other biochemical metabolites that can be identified.

Background: Almost all newborns in the US are screened at birth for multiple inborn errors of metabolism using tandem mass spectrometry. Screening tests are designed to be sufficiently sensitive so that cases are not missed. The NACB recognized a need for standard guidelines for laboratory confirmation of a positive newborn screen such that all babies would benefit from equal and optimal follow-up by confirmatory testing.Methods: A committee was formed to review available data pertaining to confirmatory testing. The committee evaluated previously published guidelines, published methodological and clinical studies, clinical case reports, and expert opinion to support optimal confirmatory testing. Grading was based on guidelines adopted from criteria derived from the US Preventive Services Task Force and on the strength of recommendations and the quality of the evidence. Three primary methods of analyte measurement were evaluated for confirmatory testing including measurement of amino acids, organic acids, and carnitine esters. The committee graded the evidence for diagnostic utility of each test for the screened conditions.Results: Ample data and experience were available to make strong recommendations for the practice of analyzing amino acids, organic acids, and acylcarnitines. Likewise, strong recommendations were made for the follow-up test menu for many disorders, particularly those with highest prevalence. Fewer data exist to determine the impact of newborn screening on patient outcomes in all but a few disorders. The guidelines also provide an assessment of developing technology that will fuel a refinement of current practice and ultimate expansion of the diseases detectable by tandem mass spectrometry.Conclusions: Guidelines are provided for optimal follow-up testing for positive newborn screens using tandem mass spectrometry. The committee regards these tests as reliable and currently optimal for follow-up testing. .

Context.—Harmonization and standardization of results among different clinical laboratories is necessary for clinical practice guidelines to be established. Objective.—To evaluate the state of the art in measuring 10 routine chemistry analytes. Design.—A specimen prepared as off-the-clot pooled sera and 4 conventionally prepared specimens were sent to participants in the College of American Pathologists Chemistry Survey. Analyte concentrations were assigned by reference measurement procedures. Participants.—Approximately 6000 clinical laboratories. Results.—For glucose, iron, potassium, and uric acid, more than 87.5% of peer groups meet the desirable bias goals based on biologic variability criteria. The remaining 6 analytes had less than 52% of peer groups that met the desirable bias criteria. Conclusions.—Routine measurement procedures for some analytes had acceptable traceability to reference systems. Conventionally prepared proficiency testing specimens were not adequately commutable with a fresh frozen specimen to be used to evaluate trueness of methods compared with a reference measurement procedure.

To the Editor: We commend Don-Wauchope et al., who assessed 11 National Academy of Clinical Biochemistry (NACB)1 laboratory medicine practice guidelines (LMPGs) with the AGREE II (Appraisal of Guidelines for Research and Evaluation II) instrument (1), and we thank Bossuyt for his accompanying editorial (2). The results of this study are helpful but are not unexpected. These LMPGs were developed over several years. The 8 nonarchived LMPGs published in the past 5 years used 4 different systems for grading and weighting practice recommendations. One system that several LMPG committees have used is a modified US Preventive Services Task Force (USPSTF) system. A second system used for 2 LMPGs was that of the American College of Cardiology and the American Heart Association. At times, NACB LMPG committees have used clinical societies' systems when these groups are primary collaborators in LMPG development, as well as significant end users of the LMPG. A third system created by the authors of 3 LMPGs focused on tumor marker tests. This unique system was developed by LMPG members who felt that considerable heterogeneity in clinical oncology practice guidelines failed to indicate a preference in any clinical society's system. Finally, authors of the most recent diabetes …

Context.—Specimens of the College of American Pathologists Neonatal Bilirubin and Chemistry surveys are inadequate for evaluating the performance of clinical laboratories in measuring serum bilirubin because they exhibit strong matrix interference. Recently published data indicate that at least 1 major clinical analyzer provided inaccurate bilirubin values for Neonatal Bilirubin Survey specimens. The composition of the specimens, bovine serum enriched with ditaurobilirubin, was responsible for the erroneous results.Objective.—This article evaluates the performance of major clinical analyzers using a survey specimen free of matrix interference.Design.—A human serum–based specimen enriched solely with unconjugated bilirubin was included in the 2003 Neonatal Bilirubin and Chemistry surveys. Its bilirubin concentration (19.4 mg/dL [332 μmol/L]) was determined by the reference method for total bilirubin.Results.—The coefficients of variation for the 4 major clinical analyzers (Dimension, Hitachi, Synchron, and Vitros) ranged from 1.9% to 3.7%. When compared to the bilirubin value measured by the reference method, mean bilirubin values of the 4 major clinical analyzers and College of American Pathologists (CAP) All Data (which refers to the grand mean and overall coefficient of variation of all method principles, all instruments according to CAP terminology) ranged from −3.5% to 5.1%. Direct bilirubin results from most field methods showed good specificity overall.Conclusion.—Human serum–based survey specimens, having their bilirubin concentrations determined by the reference method, should be included as frequently as feasible in the Neonatal Bilirubin Survey. Such specimens may be used by instrument manufacturers as standards for calibrating bilirubin methods and for assigning values to calibrators provided to instrument users. A substantial improvement in bilirubin measurements due to the reduction of systematic error is expected.

Background: The diagnosis and management of hyperbilirubinemia in the newborn requires accurate and precise bilirubin determinations. We evaluated the current status of bilirubin measurements in US laboratories by examining data submitted by laboratories participating in the College of American Pathologists (CAP) Neonatal Bilirubin (NB) and Chemistry (C) Surveys. Methods: We analyzed specimens from the CAP NB and C Surveys by the reference method for total bilirubin in three laboratories. The reference method bilirubin values were compared with bilirubin values reported by Survey participants. Results: The imprecision (CV) for all instruments combined (CAP-All Instruments) ranged from 4.7% to 5.6% at the bilirubin concentrations tested. The CVs of the four most commonly used instruments were smaller, ranging from 1.9% to 4.5%. Differences between bilirubin values by the reference method and mean values from the four most common instruments ranged from −21.6% to 10.9%. When the same specimens from the NB Surveys were used in the C Surveys, the Vitros values were strikingly different from those of the NB Surveys. The use of different methods in the NB and C Surveys coupled with the presence of a nonhuman protein base and ditaurobilirubin (DTB) in the Survey specimens accounted for the discrepant results. Conclusions: The evaluation of accuracy is impossible from the CAP Surveys because the specimens consist of bovine serum containing a mixture of unconjugated bilirubin and DTB. For the evaluation of accuracy, we recommend that Survey specimens consist of human serum enriched with unconjugated bilirubin.

We describe a case of extreme hypercholesterolemia, mediated by lipoprotein X, in a 12-year-old Caucasian female who underwent an unrelated allogenic bone marrow transplant for relapsed acute myelocytic leukemia (AML). Her post-transplant course was complicated by severe chronic graft-versus-host disease (GVHD) of the liver. Previously normal serum cholesterol and triglycerides rose to 1,122 mg/dl (29.0 mmol/L) and 1,100 mg/dl (12.4 mmol/L), respectively. Serum cholesterol appeared to be dominantly carried by lipoprotein X. Intra-hepatic cholestasis leading to reflux of bile lipoproteins into the blood stream and subsequent formation of lipoprotein X appears to be the mechanism underlying this phenomenon.

We evaluated hemoglobin (Hb) interference in the total bilirubin (TBIL) and neonatal bilirubin (NBIL) [the sum of unconjugated (Bu) and conjugated (Bc) bilirubins] methods on the Vitros 5,1 FS analyzer and assessed the reliability of the analyzer’s hemolysis and icterus index values by comparing them to measured concentrations of bilirubin and Hb in test specimens. We also compared TBIL with NBIL values in specimens from neonates 1–14 days old and obtained estimates of the extent of hemolysis in these specimens. We prepared a hemolysate from EDTA blood drawn from volunteers. After centrifugation, the cells were washed 5 times with physiologic saline, diluted with deionized water, and stored at −20 °C overnight. After thawing, the hemolysate was centrifuged to remove the stroma. The Hb concentration was measured with the CELL-DYN 4000 analyzer. We prepared a stock solution of Bu (NIST SRM 916) in pooled human serum samples from healthy volunteers as described previously (1); dilutions were made with the same pooled sera. A solution …

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.

The CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) is a NADH-dependent enzyme which catalyzes the key reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis. This highly purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity since bleaching of the [2Fe-2S] center leads to inactive enzyme. These results suggest that E3 employs a short electron-transport chain composed of both FAD and the iron-sulfur center to shuttle electrons from NADH to its acceptor. The order of electron flow, as indicated by EPR measurement with partially reduced E3, starts with hydride reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycarose (2,6-dideoxy-3-C-methyl-L-ribo-hexose), olivomycose (2,6-dideoxy-3-C-methyl-L-arabino-hexose), axenose (2,6-dideoxy-3-C-methyl-L-xylohexose), and D-evermicose (2,6-dideoxy-3-C-methyl-D-arabino-hexose) are unique, as their structures contain both two deoxy (at C-2 and C-6) and methylbranched (at C-3) moieties. A close examination of the structures of these compounds allowed us to envisage that the 2-thiobarbituric acid (TBA)-aldehyde reaction