Comparison of Bio-Rad %CDT TIA and CDTect as laboratory markers of heavy alcohol use and their relationships with gamma-glutamyltransferase.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is used as a serum marker for heavy drinking. We compared a new Bio-Rad %CDT TIA assay with the CDTect assay; we also compared both to gamma-glutamyltransferase (GGT) as markers of heavy drinking. METHODS: Serum samples of well-defined alcoholics (n = 404) and matched (age, race, and gender) social drinkers (204) from 10 clinical centers were assayed with both CDT assays. Both assays use microcolumn separation after iron saturation, followed by enzyme immunoassay (CDTect) or turbidimetric immunoassay (Bio-Rad %CDT). In the latter, CDT is expressed as a percentage of total transferrin. RESULTS: The slope and intercept [95% confidence intervals (CIs)] for linear regression of results obtained by the %CDT-TIA (as percentage) and CDTect (units/L) assays were 0.091 (0.088-0.097) and 0.70% (0.54-0.86%), respectively (S(y/x) =1.30%; r = 0.848). The areas under the ROC curves (95% CIs) for CDTect and Bio-Rad %CDT TIA were 0.89 (0.86-0.92) and 0.88 (0.85-0.91), respectively, for men (P, not significant) and 0.76 (0.72-0.80) and 0.72 (0.68-0.76) for women (P, not significant). When CDT (CDTect or Bio-Rad %CDT) was combined with GGT (either one positive), the clinical sensitivity in men was 90% for both assays, and specificities were 81% and 84%, respectively; sensitivities in women were 75% and 76%, respectively, and specificities were 87% and 91%. CONCLUSION: The new Bio-Rad %CDT TIA assay compares favorably to the widely studied CDTect assay in the detection of alcohol-use disorders.

Evaluation of novel assays in clinical chemistry: quantification of plasma total homocysteine.

BACKGROUND: There is a need for systematic evaluation of methods before their release to the market. We addressed this problem in novel homocysteine assays as part of an European Demonstration Project involving six centers in four countries. METHODS: Two immunological methods for measurement of plasma total homocysteine (P-tHcy), the fluorescence polarization immunoassay (FPIA) and the enzyme immunoassay (EIA), were compared with two comparison methods, HPLC and gas chromatography-mass spectrometry (GC-MS). All laboratories performed the following procedures: (a) familiarization; (b) determination of linearity and precision by analyzing five plasma samples with interrelated concentrations for 20 days; (c) correlation using patients' samples; and (d) assessment of long-term performance. RESULTS: Both immunological methods were linear for P-tHcy between 5 and 45 micromol/L. The intralaboratory imprecision (CV) was <5% for FPIA and <9% for EIA used with a sample processor. The bias was -2% to 3% for FPIA and 2-4% for EIA used with a sample processor. CONCLUSIONS: The immunological methods provide results with little bias compared with HPLC and GC-MS. The imprecision of the assays must be considered in the context of their intended use(s).

Isolation, cloning, and sequencing of porcine agouti exon 2 (PorAex2).

In order to isolate, clone, and sequence agouti exon 2 of the pig (Yorkshire), we used an interspecific hybridization strategy. Primers from the 5' and 3' borders of the known human agouti exon 2 sequence were used to amplify (PCR) pig agouti exon 2. Following Southern blotting using a human exon 2 internal primer to authenticate that our PCR amplified product was truly pig exon 2 (PorAex2), the fragment was cloned and sequenced. PorAex2 exhibits 79.1 and 75.7% DNA sequence and 85 and 74% deduced amino acid sequence homologies with human and mouse agouti exon 2 and agouti protein, respectively. With the isolation of PorAex2, we can now map, sequence, and clarify the modus operandi of the porcine agouti gene. The GenBank Accession number of PorAex 2 is AF018166.

Semiautomated procedures for evaluation of carbohydrate-deficient transferrin in the diagnosis of alcohol abuse.

Carbohydrate-deficient transferrin (CDT) may now be the most valuable biological marker for diagnosis of alcohol abuse. We compared the diagnostic performance of two new CDT tests, Axis %CDT turbidimetric immunoassay (TIA) and Axis %CDT HPLC, against Specialty Laboratories' isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD). Both Axis tests include one-half the concentration of trisialotransferrin isoforms in their CDT quantitation schemes. Considering an alcohol abuse prevalence of 7%, Axis %CDT TIA shows a sensitivity of 87% at 98% specificity and a positive predictive value (PPV) of 0.75; %CDT HPLC shows a sensitivity of 87% at 100% specificity for a PPV of 1, and the IEF/IB/LD shows 81% sensitivity at 94% specificity for a PPV of 0.5. All three CDT tests show the same negative predictive value (0.98). Both Axis procedures perform better than IEF/IB/LD in the diagnosis of alcohol abuse; %CDT TIA is available in several semiautomated, cost-effective formats.

Should tri-sialo-transferrins be included when calculating carbohydrate-deficient transferrin for diagnosing elevated alcohol intake?

CDT (carbohydrate-deficient transferrin) has been identified as a specific marker for chronically elevated alcohol consumption. We investigated the sensitivity and accuracy of using relative concentrations of different isotransferrins in serum for diagnosis of chronically elevated alcohol consumption. The different transferrin variants (isoforms) were quantified by HPLC. Including the trisialo-transferrin fraction into the definition of %CDT resulted in an increased accuracy in the detection of chronically elevated alcohol intake in a study among 17 heavy drinkers, 25 healthy individuals with moderate alcohol consumption and nine total abstainers. The results also suggest that desialylation of transferrin is a gradually continuing process, rather than one leading to a single end-result separating asialo-, mono- and disialo-transferrins from trisialo-, tetrasialo-, pentasialo- and higher sialo-transferrins.

Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Relative contributions of MCM1 and STE12 to transcriptional activation of a- and alpha-specific genes from Saccharomyces cerevisiae.

We have examined the relative contributions of MCM1 and STE12 to the transcription of the a-specific STE2 gene by using a 367 bp fragment from the STE2 5'-noncoding region to drive expression of a reporter lacZ gene. Mutation of the MCM1 binding site destroyed MCM1.alpha 2-mediated repression in alpha cells and dramatically reduced expression in a cells. The residual expression was highly stimulated by exposure of cells to pheromone. Likewise, the loss of STE12 function reduced lacZ expression driven by the wild-type STE2 fragment. In the absence of both MCM1 and STE12 functions, no residual expression was observed. Thus, the STE2 fragment appears to contain two distinct upstream activation sequences (UASs), one that is responsible for the majority of expression in cells not stimulated by pheromone, and one that is responsible for increased expression upon pheromone stimulation. In further support of this idea, a chemically synthesized version of the STE2 MCM1 binding site had UAS activity, but the activity was neither stimulated by pheromone nor reduced in ste12 mutants. Although transcription of alpha-specific genes also requires both MCM1 and STE12, these genes differ from a-specific genes in that they have a single, MCM1-dependent UAS system. The activity of the minimal 26 bp UAS from the alpha-specific STE3 gene was both stimulated by pheromone and reduced in ste12 mutants. These data suggest that at alpha-specific genes STE12 and MCM1 exert their effects through a single UAS.

Enhancement of complement activation and cytolysis of human IgG3 by deletion of hinge exons.

The capacity to induce complement-mediated cell lysis is greatly enhanced by truncating the hinge of IgG3 through exon deletions. This was shown by establishing five new cell lines which secreted chimeric IgG3 molecules with specificity for the hapten 4-hydroxy-3-nitrophenacetyl (NP) and having 47,45,32,15, and 0 amino acid hinge regions (the wild-type IgG3 has 62 amino acids in the hinge). Efficient complement activation and complement-mediated cell lysis did not depend on a long total hinge or on a long 'upper' hinge (the stretch from the beginning of the hinge to the first inter-heavy chain S-S bond). On the contrary, the mutant having a 15 amino acid hinge element was up to 10 times more efficient in complement lysis than the wild type. Thus the complement-activation potential appeared to be down-regulated in the wild type. On the other hand, the mutant lacking the hinge altogether did not activate complement or induce complement-mediated cytolysis. These findings have to be taken into account when antibodies are designed for human therapy.

C1q binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids.

We have constructed four different deletion mutants of a chimeric mouse-human IgG3 anti-(4-hydroxy-3-nitrophenyl)acetyl/(5-iodo-4 hydroxy-3 nitrophenyl) acetyl (NP/NIP) antibody lacking one or more of the four exons coding for the hinge region. The mutant variants all retained intact hinge region epitopes since they all reacted with IgG3 hinge-specific antibodies. Surprisingly, all the deletion mutants bound C1q equally well or even better than the wild type. Thus the high C1q binding activity of IgG3 compared to IgG1 is apparently not due to the total length of the IgG3 hinge, which is 62 amino acids, nor is it due to the length of the upper hinge which is the stretch from the end of CH1 to the first inter-heavy chain disulfide bond.

Semicontinuous diffusion fermentation of fodder beets for fuel ethanol and cubed protein feed production.

A novel, semicontinuous diffusion fermentation system was used to produce fuel ethanol and a cubed protein feed (CPF) from fodder beets at an intermediate scale. In the process, fodder beet cubes were augered diagonally upward against a flow of 0.26N H(2)SO(4) and yeast in a tubular fermentor. Exiting one end of the fermentor was CPF, while fermented beer [6-9% (v/v) ethanol] exited the other end. Retention times for beer and CPF were 264 and 72 h, respectively. Contamination was controlled by maintaining the fermentation pH between 2.1 and 2.6 using H(2)SO(4). Production costs for a greatly scaled-up (times 1400) conceptual version of this system (using a continuous rather than a semicontinuous processing mode) were projected by calculation to be $0.529/L for 95% ethanol (net of a $0.112/L credit for CPF). The calculated energy balance (energy output-energy input ratio) was estimated to be 3.04. In calculating the energy balance, the output energy of the CPF and input energy for growing the fodder beets were not included. A design for the scaled-up plant is provided.

Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.

Effects of inoculum size on solid-phase fermentation of fodder beets for fuel ethanol production.

This fuel ethanol study examined the effects of Saccharomyces cerevisiae inoculum size on solid-phase fermentation of fodder beet pulp. A 5% inoculum (wt/wt) resulted in rapid yeast and ethanol (9.1% [vol/vol]) production. Higher inocula showed no advantages. Lower inocula resulted in lowered final yeast populations and increased fermentation times.

Intermediate-scale, semicontinuous solid-phase fermentation process for production of fuel ethanol from sweet sorghum.

A novel, semicontinuous solid-phase fermentation system was used to produce fuel ethanol from sweet sorghum. The process was at an intermediate scale. In the process, dried and shredded sweet sorghum was rehydrated to 70% moisture, acidified to pH 2.0 to 3.0, and either pasteurized (12 h at 70 to 80 degrees C) or not pasteurized before spray inoculation with a broth culture of Saccharomyces cerevisiae. Fermented pulp exited the semicontinuous fermentor after a retention time of 72 h and contained approximately 6% (vol/vol) ethanol. Ethanol yields from dry sweet sorghum were 176 to 179 liters/10 kg (85% of theoretical). Production costs for a greatly scaled-up (x1,400) conceptual version of this system were projected by calculation to average $0.47/liter for 95% ethanol. The calculated energy balance (energy output/energy input ratio) was estimated to be 1.05 when pasteurization was included and 1.31 when pasteurization was omitted. In calculating the energy balances, the output energy of the protein feed byproduct and the input energy for growing the sweet sorghum were not considered. A design for the scaled-up plant (farm scale) is provided.

A continuous, farm-scale, solid-phase fermentation process for fuel ethanol and protein feed production from fodder beets.

Fuel ethanol (95%) was produced from fodder beets in two farm-scale processes. In the first process, involving conventional submerged fermentation of the fodder beets in a mash, ethanol and a feed (PF) rich in protein, fat, and fiber were produced. Ethanol yields of 70 L/metric ton (7 gal/ton) were obtained; however, resulting beers had low ethanol concentrations [3-5% (v/v)]. The high viscosity of medium and low sugar, beet mashes caused mixing problems which prevented any further increase of beet sugar in the mash. The severely limited the maximum attainable ethanol concentration during fermentation, thereby making the beer costly to distill into fuel ethanol and the process energy inefficient. In order to achieve distillably worthwhile ethanol concentrations of 8-10% (v/v), we developed and tested a solid-phase fermentation process (continuous). In preliminary trials, this system produced fermented pulp with over 8% (v/v) ethanol corresponding to an ethanol yield of 87 L/metric ton (21 gal/ton). Production costs with this novel process are $0.47/L ($1.77/gal) and the energy balance is 2.11. These preliminary cost estimates indicate that fodder beets are potentially competitive with corn as an ethanol feedstock. Additional research, however, is warranted to more precisely refine individual costs, energy balances and the actual value of the PF.

Metabolism of various carbon sources by Azospirillum brasilense.

Azospirillum brasilense Sp7 and two mutants were examined for 19 carbon metabolism enzymes. The results indicate that this nitrogen fixer uses the Entner-Doudoroff pathway for gluconate dissimilation, lacks a catabolic but has an anabolic Embden-Meyerhof-Parnas hexosephosphate pathway, has amphibolic triosephosphate enzymes, lacks a hexose monophosphate shunt, and has lactate dehydrogenase, malate dehydrogenase, and glycerokinase. The mutants are severely deficient in phosphoglycerate and pyruvate kinase and also have somewhat reduced levels of other carbon enzymes.

Fuel ethanol and high protein feed from corn and corn-whey mixtures in a farm-scale plant.

Distiller's wet grain (DWG) and 95% ethanol were produced from corn in a farm-scale process involving batch cooking-fermentation and continuous distillation-centrifugation. The energy balance was 2.26 and the cost was $1.86/gal (1981 cost). To improve the energy balance and reduce costs, various modifications were made in the plant. The first change, back-end (after liquefaction) serial recycling of stillage supernatant at 20 and 40% strengths, produced beers with 0.2 and 0.4% (v/v) more ethanol, respectively, than without recycling. This increased the energy balance by 0.22-0.43 units and reduced costs by $0.07-$0.10/gal. The DWGs from back-end recycling had increased fat. The second change, increasing the starch content from 17-19% to 27.5%, increased the ethanol in the beer from 10.5-14.9% at a cost saving of $0.41/gal. The energy balance increased by 1.08 units. No significant change was seen in DWG composition. The third change, using continuous cascade rather than batch fermentation, permitted batch-levels of ethanol (10%) in the beer but only at low dilution rates. Both the cost and energy balance were decreased slightly. The DWG composition remained constant. The last change, replacing part of the corn and all of the tap water in the mash with whole whey and using Kluyveromyces fragilis instead of Saccharomyces cerevisiae during fermentation, resulted in an energy balance increase of 0.16 units and a $0.27/gal cost reduction. Here, 10% ethanolic beers were produced and the DWGs showed increased protein and fat. Recommendations for farm-scale plants are provided.

Farm-scale production of fuel ethanol and wet grain from corn in a batch process.

The batch production of fuel grade ethanol and distillers' wet grain (wet solids) in a farm-scale process (1240-15,580 L/batch) is described. The employs yeast fermentation of amylase-treated corn mash and a two-stage distillation. Primary emphasis in this study was on the cooking, fermentation, and centrifugation steps. Without recycling, fermentation of the mash yield beers with 10.0-10.5% ethanol. Recycling of stillage supernatant at full, 75, or 50% strengths produced enriched mashes that after 48-h fermentation yielded beers with 5-;14% more ethanol. Recycling twice with full-strength supernatant at pH 7.0 increased the ethanol yield in the final beer 16.5%; however, the time to complete the final fermentation was extended form 48 to 72 h and salt buildup occurred. By recycling at pH 5.4, it was possible to avoid rapids salt buildup and obtain beers with 10.3-10.5% ethanol. Recycling resulted in increased levels of glucose, starch, crude protein, and fat in the beer and a reduced moisture content while the wet solids showed an increased starch content. Centrifugation after cooking or fermentation yield in the subsequently produced beer. Fermentation of a volume-resorted mash supernatant gave a beer with only 9.25% ethanol. Mash wet solids varied somewhat chemically from beer and stillage solids. An economic and energy balance analysis of various modes of plant operation are provided and plant considerations are suggested.