Plasma protein contents determined by Fourier-transform infrared spectrometry.

BACKGROUND: Fourier-transform infrared (FT-IR) spectrometry has been used to measure small molecules in plasma. We wished to extend this use to measurement of plasma proteins. METHODS: We analyzed plasma proteins, glucose, lactate, and urea in 49 blood samples from 35 healthy subjects and 14 patients. For determining the concentration of each biomolecule, the method used the following steps: (a) The biomolecule was sought for which the correlation between spectral range areas of plasma FT-IR spectra and concentrations determined by comparison method was greatest. (b) The IR absorption of the biomolecule at the most characteristic spectral range was calculated by analyzing pure samples of known concentrations. (c) The plasma concentration of the biomolecule was determined using the FT-IR absorption of the pure compound and the integration value obtained for the plasma FT-IR spectra. (d) The spectral contribution of the biomolecule was subtracted from the plasma FT-IR spectra, and the resulting spectra were saved for further analyses. (e) The same method was then applied to determining the concentrations of other biomolecules by sequentially comparing the resulting FT-IR spectra. RESULTS: Results agreed with those obtained by clinical methods for the following biomolecules when analyzed in the following order: albumin, glucose, fibrinogen, IgG(2), lactate, IgG(1), alpha(1)-antitrypsin, alpha(2)-macroglobulin, transferrin, apolipoprotein (Apo)-A(1), urea, Apo-B, IgM, Apo-C(3), IgA, IgG(4), IgG(3), IgD, haptoglobin, and alpha(1)-acid glycoprotein. CONCLUSION: FT-IR spectrometry is a useful tool for determining concentrations of several plasma biomolecules.

Analysis of the polymorphism of the tumour necrosis factor (TNF) gene and promoter and of circulating TNF-alpha levels in heart-transplant patients suffering or not suffering from severe rejection.

Plasma TNF-alpha levels are generally higher in heart-graft patients who experience a rejection episode than in those who do not. Because the TNF gene and its promoter are polymorphic, we studied the relationships between genetic variability at the TNF locus, the occurrence of graft rejection and TNF-alpha plasma levels in 62 heart-transplant patients in order to investigate inter-individual differences in plasma TNF-alpha levels after allogeneic stimulation. TNF-alpha was immunoenzymatically measured in blood specimens collected on the same day as endomyocardial biopsy. After PCR amplification of DNA, NcoI and AspHI polymorphisms were characterized by their restriction profiles, TNFa microsatellites by electrophoretic separation on acrylamide and the promoter region by sequencing. Plasma levels and molecular genetic results were compared to the grade of heart graft rejection established according to pathological criteria. In our study, allograft rejection was associated neither with NcoI or AspHI polymorphism nor with nucleotide changes in the TNF-A promote. We observed low TNF-alpha levels in n1/n1 homozygous patients and in subjects with G-->A at position--308 of the promoter sequence. Concerning the polymorphism of the TNFa microsatellite, our results might suggest an association with graft rejection but we have to be very careful in drawing conclusions because of the small size of the sample.

Phosphoserine aminotransferase, the second step-catalyzing enzyme for serine biosynthesis.

As a step toward analyzing the serine biosynthetic pathway in mammals, we have studied the properties of phosphoserine aminotransferase, the second step-catalyzing enzyme. The K(m) values for 3-phosphohydroxypyruvate and L-phosphoserine are 5 and 35 microM, respectively, and those for glutamate and alpha-ketoglutarate are 1.2 and 0.8 mM, respectively. The product inhibition studies strengthened the support for a ping-pong mechanism and allowed evaluation of Ki values for the four substrates. The equilibrium constant evaluated from the kinetic parameters is approximately 40. Additionally, some physical properties relative to the bound coenzyme and the secondary structure were investigated. The results are consistent with a structural relationship between the Escherichia coli enzyme and the mammalian enzyme. The mammalian enzyme has specific kinetic parameters, the determination of which is a prerequisite to analyzing the serine biosynthetic pathway in mammals.

First characterization of the phosphonoacetaldehyde hydrolase gene of Pseudomonas aeruginosa.

The phnX gene encoding the phosphonoacetaldehyde hydrolase (phosphonatase) from the Gram-negative bacterium Pseudomonas aeruginosa A237 has been cloned and its sequence determined. The open reading frame consists of 825 nucleotides specifying a protein of 275 amino acid residues corresponding to a predicted molecular weight of 29929. The deduced amino acid sequence of PhnX did not share significant amino acid sequence similarity with any other polypeptide. Expression of the phosphonoacetaldehyde hydrolase coding sequence in Escherichia coli under control of the E. coli tac promoter resulted in the production of enzymatically active protein with an affinity constant similar to that of the phosphonoacetaldehyde hydrolase purified from P. aeruginosa A237. This is the first nucleic sequence report of the phosphonoacetaldehyde hydrolase, an enzyme involved in the carbon-phosphorus bond cleavage.

Evaluation of plasma levels of tumour necrosis factor alpha and interleukin-6 as rejection markers in a cohort of 142 heart-grafted patients followed by endomyocardial biopsy.

The rejection reaction after cell or organ transplantation has to be detected as early as possible in order to conduct optimal immunosuppressive treatment. Among the numerous events leading to rejection, cytokine production, especially of tumour necrosis factor alpha, is particularly important. Interleukin-6 and tumour necrosis factor alpha were investigated in 142 heart-grafted patients in order to define an early peripheral non-invasive marker of an acute rejection that could fit well with myocardial biopsy results. Cytokines were immunoenzymatically measured in blood specimens collected on the day of the endomyocardial biopsy. The values were compared to the grade of heart graft rejection established according to pathological criteria. Plasma interleukin-6 and especially tumour necrosis factor alpha determined on the day of the rejection diagnosis were significantly increased in the patient sample with moderate or severe rejection when compared with mean values of interleukin-6 and tumour necrosis factor alpha in the patient sample without rejection or with mild rejection (P = 0.04 and 0.001 respectively). Because high levels of tumour necrosis factor alpha may appear before histological signs, this biological marker could be useful in the follow-up of heart-grafted patients.

Laryngeal and oropharyngeal cancer, and alcohol dehydrogenase 3 and glutathione S-transferase M1 polymorphisms.

In this study the GSTmu phenotype and ADH genotype at the ADH3 locus were investigated in a group of 39 alcoholic men with upper respiratory/digestive tract cancer: 21 with oropharyngeal cancer and 18 with laryngeal cancer. The results are compared with those of a control group of 37 alcoholic men without alcohol-related medical complications. Of the control subjects, 48% were found to be GSTmu deficient [GSTmu(-)] and 19% carried the ADH(3)1/ADH(3)1 genotype. In the laryngeal cancer patients, a significantly elevated frequency of both the GSTmu(-) (78%) and ADH(3)1/ADH(3)1 genotype (56%) was observed, relative to the control group. On the basis of this result, the risk of laryngeal cancer associated with the GSTmu(-) and ADH(3)1/ADH(3)1 genotypic combination within the population of alcoholics was estimated to be 12.9 with a 95% confidence interval of 1.8-92 (P < 0.01) relative to alcoholic individuals who have GSTmu [GSTmu(+)] and are not ADH(3)1/ADH(3)1. Thus, alcoholics who are GSTmu(-) and ADH(3)1/ADH(3)1 have at least an 80% greater risk of developing laryngeal cancer than alcoholics who are GSTmu(+) and who are not ADH(3)1/ADH(3)1. In addition, the oropharyngeal cancer patients had excess frequencies of both GSTmu(-) (62%) and ADH(3)1/ADH(3)1 (43%) relative to the control group, but these excess frequencies were not statistically significant. The GSTmu(-) and ADH(3)1/ADH(3)1 genotypic combination may be a constitutional risk factor for laryngeal cancer among alcoholics.

Plasma cytokines in graft vs host disease and complications following bone marrow transplantation.

A cohort of 201 autologous or allogeneic bone marrow transplanted (BMT) patients were included for studying the evolution of circulating tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) by means of repeated plasma determinations. IL-6 levels were high during major transplant-related complications (TRC) or severe graft vs host disease (GVHD). High levels of TNF-alpha seemed to be associated with chronic GVHD but not with acute GVHD or TRC.

The frequency of the mitochondrial aldehyde dehydrogenase I2 (atypical) allele in Caucasian, Oriental and African black populations determined by the restriction profile of PCR-amplified DNA.

The aldehyde dehydrogenase I (ALDH I) gene codes for a mitochondrial enzyme which plays a major role in hepatic alcohol detoxication. It has been related to alcohol flushing in Orientals bearing the atypical ALDH I2 gene. The variant protein results from a lysine for glutamate substitution at position 487 (G-->A change in exon 12). A procedure for ALDH I2 detection consisting in a differentiation between the 'atypical' allele and the 'wild' allele has been improved through PCR and subsequent MboII digestion. Blood samples collected on anticoagulant or directly absorbed on blotting paper were used for DNA amplification in the presence of two specific oligonucleotidic primers, each one able to incorporate a restriction site in the amplimer. After MboII digestion, PCR products were separated by polyacrylamide gel electrophoresis and then visualized with ethidium bromide. This technique permits a rapid and non-radioactive detection of atypical ALDH I2 on a PCR product without the use of allele specific oligonucleotides. It was applied to the study of ALDH I2 allele frequency in random population samples of three ethnic groups: Caucasians, Orientals and African blacks.

Identification of a new apolipoprotein E variant (E2 Arg142-->Leu) in type III hyperlipidemia.

A new rare apolipoprotein E mutant was identified as we were investigating the apolipoprotein E genotype of patients with type III hyperlipidemia (HLP III). The unusual DNA restriction fragment length polymorphism profile and then the sequence analysis of a PCR amplified fragment of the proband's apo E gene revealed a simple base substitution (G-->T) at nucleotide 3836. This mutation leads to the replacement of arginine by leucine at position 142 of the mature protein. The proband carried the mutant allele at the heterozygous status with an epsilon 3 allele. Subsequently, analysis of the proband's father's apo E gene showed that same mutated allele associated with an epsilon 2 allele. The two subjects presented a dysbetalipoproteinemia in which this new apo E variant could be implicated.

Genetic polymorphism of apolipoprotein E in Caucasian alcoholic cirrhotics.

Steatosis is the most common although inconstant hepatic lesion induced by chronic alcohol consumption. Alcoholic cirrhosis is found in only 20 to 30% of chronic alcohol misusers, which points to the influence of both environmental and genetic factors. Apolipoprotein E (apo E) presents an important polymorphism with three common alleles, epsilon 4, epsilon 3 and epsilon 2. Its modulation role on triglyceride-rich lipoproteins and cholesterol-containing lipoproteins is linked with its interaction with cellular specific receptors. This work aims at studying a possible correlation between apo E polymorphism and alcoholic cirrhosis. The three common alleles were identified by enzymatic amplification (PCR) of genomic DNA from blood samples and analysis of restriction profiles. The distribution of alleles and genotypes was performed in 35 Caucasian cirrhotic patients (medium age 57 years) and compared with the usual distribution of apo E phenotypes in European Caucasian populations. The results show lower epsilon 4 and epsilon 2 allele frequencies and higher epsilon 3 allele frequency in Caucasian alcoholic cirrhotics.

Application of PCR site-directed mutagenesis for a rapid and accurate detection of mutation 3500 (Arg-->Gln) of human apolipoprotein B-100.

A rapid detection of the Arg3500-->Gln mutation of human apolipoprotein B-100 is of particular interest because of its prevalence in familial forms of hypercholesterolemia. A simple procedure, based on amplification by polymerase chain reaction (PCR) and NlaIII endonuclease restriction cleavage, allows this diagnosis without ambiguity. By using two oligonucleotide primers carrying one mismatch each, two permanent restriction sites were generated in the normal allele, while one of them disappeared in the mutant allele. Thus, the two alleles can be differentiated by their specific N/aIII restriction profile.

[Pathology of the human apolipoprotein E gene]. / Pathologie du gène de l'apolipoprotéine E humaine.

Apolipoprotein E (apo E) is a polymorphic glycoprotein that plays an essential part in the binding to receptors for the uptake of chylomicrons and VLDL remnants and of LDL. The three major isoforms are E3 (Cys112/Arg158), E4 (Arg112/Arg158) and E2 (Cys112/Cys158). The apo E genetic variation has a great impact. In most of type III familial hyperlipoproteinemias (HLP), E2 is implicated at the homozygote status. In other cases, rare alleles are directly responsible for dominant type III HLP. Apo E polymorphism is an essential determinant in the interindividual variations of lipids in healthy subjects in various populations. Its influence can be significant on the efficacy of nutritional or therapeutic interventions. The allele epsilon 4 appears to be associated with an increased risk of premature atherosclerosis. Recently, epsilon 4 was demonstrated to be associated with an early Alzheimer's disease onset. Apo E polymorphism contributes to the lipid disorders in diabetes and obesity. The analysis of apo E polymorphism can be carried out with two conceptually different approaches. The first one is based on the separation of plasma isoforms of the protein by isoelectric focusing or bidimensional electrophoresis. The other one consists in the application of molecular biology techniques (PCR and endonuclease restriction profiles) for a detection of the common alleles and of several rare alleles, avoiding the possible errors of the phenotyping technique of the apo E protein. The application of genetic engineering allows a better understanding of the role played by apo E towards its receptors and in other molecular interactions which are not well known up to now.

Common and rare genotypes of human apolipoprotein E determined by specific restriction profiles of polymerase chain reaction-amplified DNA.

The three common isoforms of human apolipoprotein E (apo E) differ at positions 112 and 158 and are named E3, E4, and E2 according to phenotyping by isoelectric focusing (IEF). The polymerase chain reaction (PCR) method allows the detection of common and several rare allelic apo E variants not detected by IEF. We propose a genotyping procedure for apo E that characterizes a given allele on the basis of amplification of specific sequences of the gene followed by the action of restriction endonucleases. When the nucleotide change does not lead to a restriction site, PCR-directed mutagenesis creates the discriminant site, and the differentiation of the three common alleles and five rare variants is possible. We present here profiles of common alleles and of three rare alleles, Weisgraber [Cys112/Asp127/Cys158], Christchurch [Cys112/Ser136/Arg158], and a new rare variant [Cys112/Leu142/Cys158].

Effect of the phosphonic analogue of tyrosine on tyrosine iodination.

Depositing ofDL-1-amino-2-(p-hydroxyphenyl)-ethylphosphonic acid (Tyr-P) on the chicken embryo induced a dose dependent decrease of the iodine uptake by the embryonic thyroid. Tyr-P interfered on iodination of tyrosine when tested with hog thyroid peroxidase (TPO) and with bovine lactoperoxidase (LPO); the analogue was recognized by the two enzymes but its affinity for TPO and LPO was respectively 3 and 7 fold higher compared with that of the natural substrate, suggesting that Tyr-P may act as an iodine trap.

Action of the phosphonic analogue of tyrosine and of other tyrosine derivatives on rat liver tyrosine aminotransferase.

Tyrosine transamination has been investigatedin vitro with a preparation of rat liver tyrosine aminotransferase in the presence of several structural derivatives of the substrate, including the phosphonic analogue. The transamination by tyrosine aminotransferase (TAT) needs the presence in the substrate molecule of free amino and carboxylic groups, a three-carbon aliphatic chain, a para-phenolic hydroxylic function and aL-configuration. Some tyrosine analogues can markedly disturb the Tyr-TAT association: the chief structural modifications are (i) the removal of the free amine function in a compound still possessing a para-hydroxylic and a carboxylic group, (ii) the change of the carboxylic function by another acidic group, especially a phosphonic one, (iii) a disubstitution in positions 3 and 5. In every situation, the presence of a parahydroxylic group is compulsory to observe an inhibitory effect.

Polymorphism of class I alcohol dehydrogenase in French, Vietnamese and Niger populations: genotyping by PCR amplification and RFLP analysis on dried blood spots.

The polymorphism of human alcohol dehydrogenase (ADH) can contribute to the explanation of the important ethnic differences towards alcohol metabolism. Its assessment at the genomic DNA level with a procedure, excluding labelled probes, consisting of PCR (Polymerase chain reaction) amplification on dried blood spots and analysis of allele-specific RFLP (Restriction fragment length polymorphism) profiles, is well adapted to extensive studies in population samples. It can emphasize the importance of ADH as a genetic marker of population. Three ethnic groups (French Caucasians, Vietnamese Orientals, Black Africans from Niger) were studied. ADH2 and ADH3 genotypes were in equilibrium according to the Hardy-Weinberg law. Important differences were noted in the distribution of ADH2 and ADH3 alleles.

[Optimization of a spectrophotometry assay of total and oxidized blood glutathione: comparison with a fluorimetric method]. / Optimisation du dosage spectrophotométrique du glutathion sanguin total et oxydé: comparaison avec une méthode fluorimétrique.

We developed a method for the enzymatic assay of glutathione which is easy to practice, rapid, specific, based on the reaction of the thiol group of glutathione with dithiobis-nitrobenzoic acid after the action of glutathione reductase in the presence of NADPH. This spectrophotometric technique allowed, on the one hand, the determination of total glutathione and on the other hand, that of oxidized glutathione (disulfide), after the blockage of reduced glutathione by 2-vinyl-pyridine. The improvements of the assay of blood glutathione concerned the sample preparation, the reaction sensitivity, thanks to a better definition of the optimal pH and a reduction ot the blockage time by 2-vinyl-pyridine in well defined operating conditions. We compared the performances of our technique with a fluorimetric method. We used our method for the determination of total and oxidized blood glutathione in a control population.

Allosteric regulation of phosphonoacetaldehyde hydrolase by n-butylphosphonic acid.

The effect of n-butylphosphonic acid on the activity of phosphonoacetaldehyde hydrolase from Pseudomonas aeruginosa was investigated: at low concentrations this compound appeared as an activator of the enzyme activity, whereas at higher concentrations it exhibited inhibitory properties. The experimental results were modelled according to an allosteric model involving two different classes of sites for n-butylphosphonic acid.