ABSTRACT
Time-resolved Fourier transform infrared spectroscopy provides evidence of non-native antiparallel beta-sheet structural elements in the folding intermediates of alpha-lactalbumin.
Subject(s)
Lactalbumin/chemistry , Lactalbumin/metabolism , Protein Folding , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Protein Denaturation , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared , ThermodynamicsABSTRACT
A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the lambda Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel beta-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20-95 degrees C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal beta-strand, followed by the unfolding of the alpha-helices in a second step, and the third step comprises the reorganization of the remaining beta-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two beta-strands at the dimer interface.
Subject(s)
Bacteriophage lambda/chemistry , DNA-Binding Proteins , Protein Folding , Repressor Proteins/chemistry , Spectrophotometry, Infrared/methods , Dimerization , Viral Proteins , Viral Regulatory and Accessory ProteinsABSTRACT
Previous studies on intact cells have shown that bryostatin 1 (Bryo 1) induces significant alterations in the membranes of WSU-CLL cells (a drug-resistant B-CLL cell line), changes which may play an important role in the mechanism of reduced drug resistance of B-CLL cells to 2-chlorodeoxyadenosine (2-CdA). However, it is not clear whether the plasma membranes or the mitochondria, or both are involved; nor is it known which of these two targets is more important for regaining the cells former drug sensitivity. For the present study, we treated WSU-CLL cells with Bryo 1, isolated plasma membranes and mitochondria, and then subjected the purified fractions to infrared (IR) spectroscopic and chromatographic analyses. IR spectroscopy revealed a decreased glycosylation of both plasma membranes and mitochondria in Bryo 1-treated cells compared to untreated cells. The amount of lipid relative to protein was increased in both types of membranes, but considerably more enhanced in the plasma membrane fraction of the Bryo 1-treated cells than in mitochondria. Quantitative lipid analysis by thin layer chromatography also revealed that Bryo 1 treatment significantly increased the phospholipid content in plasma membranes, whereas the lipids in the mitochondria remained essentially unchanged. Changes in lipid composition were quite dramatic for plasma membranes where phosphatidylcholines were decreased by 50%, phosphatidylethanolamines doubled and sphingomyelins increased five-fold compared to the lipid composition in plasma membranes of untreated cells. In addition, the IR spectroscopic analysis provided evidence for an increased plasma membrane fluidity in Bryo 1-treated cells, whereas the fluidity of the mitochondria remained essentially unchanged; marker bands indicating mitochondrial DNA decreased upon Bryo 1 treatment. These results suggest that Bryo 1 increases the sensitivity of WSU-CLL cells to chemotherapeutic agents such as 2-CdA by action on two cell targets: (1) introduction of significant changes in plasma membrane permeability or fluidity through modifications in lipid content and composition as well as by reducing the surface glycosylation; (2) introduction of changes in lipid and DNA content of the mitochondria. Small alterations in the lipid composition of the mitochondria may provide the conditions for an altered proton gradient and transmembrane potential leading to apoptosis and decreased cell survival.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Membrane/drug effects , Cell Membrane/pathology , Lactones/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Antineoplastic Agents/therapeutic use , Bryostatins , Cell Membrane/chemistry , Drug Resistance, Neoplasm , Humans , Lactones/therapeutic use , Macrolides , Membrane Lipids/chemistry , Membrane Proteins/chemistry , Mitochondria/drug effects , Mitochondria/pathology , Mitochondria/ultrastructure , Spectroscopy, Fourier Transform Infrared , Tumor Cells, CulturedABSTRACT
The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of "short" bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8 degrees C). Based on various spectroscopic data, on the known three-dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238-248.
Subject(s)
Adenylate Kinase/chemistry , Adenylate Kinase/metabolism , Models, Molecular , Mycobacterium tuberculosis/enzymology , Adenylate Kinase/genetics , Adenylate Kinase/isolation & purification , Amino Acid Sequence , Animals , Circular Dichroism , Cloning, Molecular , Enzyme Stability , Kinetics , Molecular Sequence Data , Molecular Weight , Mycobacterium tuberculosis/genetics , Nuclear Magnetic Resonance, Biomolecular , Protein Denaturation , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spectrophotometry, Infrared , Temperature , Tosylphenylalanyl Chloromethyl Ketone , Trypsin/metabolismABSTRACT
Fourier transform infrared (FTIR) microspectroscopy is emerging as a promising new tool for histopathological investigations of tissue histochemistry. This study was designed to assess whether changes in tissue biochemistry induced by well-differentiated and poorly differentiated oral/oropharyngeal squamous cell carcinoma (SCC) can be detected by infrared spectroscopy. The biopsies analyzed were each proven SCC positive and compared with tissue taken from the contralateral normal site. Individual infrared spectra, recorded from specific tissue areas, were correlated with histopathological structures normally found in the oral mucosa. Infrared mapping of these areas allows the generation of biochemical images of molecular structures such as lipids, sugars, and proteins. The visualization of DNA and tissue structures containing keratin (well expressed in all epithelia) reveals distinct differences between normal and SCC-positive biopsies. Bivariate histogram analysis of cell components (e.g., DNA and keratin) indicated that cancer cells produce a relatively homogeneous and clearly abnormal cell biochemistry, whereas differentiated epithelial cells present a very heterogeneous distribution of cellular components. Using these features, tissue containing abnormal or cancer cells can easily be distinguished from normal epithelial structures. The abnormal keratin distribution in poorly differentiated SCC and in keratin pearls (present only in well-differentiated SCC) offers insight into the process of malignant tissue transformation in squamous epithelium. Applying infrared microspectroscopy in combination with bivariate statistics to histopathological tissue thin sections provides a potential diagnostic tool for detection of cell changes in epithelial cancers.
Subject(s)
Carcinoma, Squamous Cell/pathology , Keratins/metabolism , Mouth Neoplasms/pathology , Pharyngeal Neoplasms/pathology , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/metabolism , DNA/analysis , Female , Humans , Keratins/analysis , Male , Middle Aged , Mouth Mucosa/chemistry , Mouth Mucosa/cytology , Mouth Neoplasms/metabolism , Pharyngeal Neoplasms/metabolism , Spectroscopy, Fourier Transform InfraredABSTRACT
We introduce near-IR spectroscopy as an ancillary tool for monitoring structural changes of proteins in aqueous solution using ribonuclease A (RNase A) as a model protein. The thermal unfolding of RNase A results in clear spectral changes in the near-IR and the mid-IR regions. In the near-IR the most pronounced changes are observed in the spectral region between 4820 and 4940 cm-1. The strong N-H combination band found at 4867 cm-1 in the spectrum of native RNase A shifts to 4878 cm-1 upon thermal unfolding. Hydrogen-deuterium exchange experiments that validate the N-H character of this mode can also be used to estimate the number of unexchanged amide protons after exposure to D2O. The transition profiles and temperatures derived from the temperature dependence of the N-H combination mode were found to be practically identical with those derived from the temperature dependence of the C = O amide I band in the mid-IR region, demonstrating that the near-IR region can be used as a conformation-sensitive monitor for the thermally induced unfolding of proteins in H2O solution. A 2-dimensional correlation analysis was applied to the mid-IR and near-IR spectra of RNase A to establish correlations between IR bands in both regions. The correlation analysis demonstrates that the thermal unfolding of RNase A is not a completely cooperative process; rather it begins with some changes in beta-sheet structure, followed by the loss of alpha-helical structures, and then ending with the unfolding of the remaining beta-sheets.
Subject(s)
Ribonuclease, Pancreatic/chemistry , Spectrophotometry, Ultraviolet/methods , Amides/chemistry , Deuterium Oxide , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Protons , Solutions , Thermodynamics , WaterABSTRACT
Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the ternary system ReLPS/DMPEd54/PLL(500) showed the features of a phase segregation phenomenon not affecting the transition temperatures of the pure lipid components, the ternary system composed of ReLPS/DMPEd54 and OmpF exhibited phase transition curves that were characterized by an unspecific (DMPEd54/OmpF) and a strong and unique (ReLPS/OmpF) type of lipid-protein interaction. Furthermore, semiquantitative estimations supported the supposition that OmpF might be able to induce bilayer asymmetry in preformed symmetrical ReLPS/DMPEd54 vesicles.
Subject(s)
Dimyristoylphosphatidylcholine/chemistry , Lipopolysaccharides/chemistry , Liposomes/chemistry , Phosphatidylethanolamines/chemistry , Phospholipids/chemistry , Polylysine/chemistry , Porins/chemistry , Bacterial Proteins/chemistry , Deuterium , Escherichia coli , Models, Theoretical , Molecular Conformation , Mutation , Protein Conformation , Salmonella/genetics , Spectroscopy, Fourier Transform Infrared/methods , Temperature , ThermodynamicsABSTRACT
Infrared (IR) spectroscopy was used to compare the drug resistance mechanism of cells from chronic lymphocytic leukemia (CLL) patients with that of WSU-CLL cells. Bryostatin 1 (Bryo 1), a macrocyclic lactone and protein kinase C activator, was used to render WSU-CLL cells more susceptible to 2-chlorodeoxyadenosine (2-CdA). The IR spectroscopic analysis revealed some changes in protein and DNA content in Bryo 1-treated WSU-CLL cells, however, the most significant alterations were observed in the membrane lipids, which resemble those found between 2-CdA-sensitive and 2-CdA-resistant cells from CLL patients. In addition, Bryo 1 treatment induced WSU-CLL cells to become CD11c, CD25 and tartrate-resistant acid phosphatase-positive, specific markers for hairy cell leukemia, a disease exquisitely sensitive to 2-CdA. Our results suggest that 2-CdA-sensitive CLL cells have cellular characteristics resembling the hairy cell stage. The similarity between the membrane lipids in 2-CdA-sensitive CLL cells and the Bryo 1-treated WSU-CLL cell line supports the suggestion that membrane lipid alteration might be an important step in the drug resistance mechanism of CLL cells.
Subject(s)
Cladribine/pharmacology , Lactones/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Adult , Aged , Antineoplastic Agents/pharmacology , Bryostatins , Drug Resistance , Female , Humans , Lymphocytes/drug effects , Macrolides , Male , Middle Aged , Spectrophotometry, Infrared , Tumor Cells, CulturedABSTRACT
A new class of outer membrane lipid (OML) was isolated from the oral spirochete Treponema denticola strain ATCC 33521 using a phenol/chloroform/light petroleum procedure normally applied for lipopolysaccharide extraction. In addition to chemical analysis, Fourier transform infrared (FTIR) spectroscopy was applied to compare the biophysical properties of OML with lipopolysaccharides (LPS) and lipoteichoic acids (LTA). Isolated OML fractions represent 1.4% of the total dry cell weight, are about 4 kDa in size, and contain 6% amino sugars, 8% neutral sugars, 14% phosphate, 35% carbazol-positive compounds, and 11% fatty acids (containing iso- and anteiso-fatty acyl chains). Rare for outer membrane lipids, OML contains no significant amount of 3-deoxy-D-manno-octulosonic acids, heptoses, and beta-hydroxy fatty acids. The fatty acyl chain composition, being similar to that of the cytoplasmic membrane, is quite heterogeneous with anteiso-pentadecanoic acid (12%), palmitic acid (51%), and iso-palmitic acid (19%) as the predominant fatty acids present. Findings of a glycerol-hexose unit and two glycerol-hexadecanoic acid fragments indicate a glycolipid membrane anchor typically found in LTA. There was also no evidence for the presence of a sphingosine-based lipid structure. The results of FTIR measurements strongly suggest that the reconstituted lipid forms normal bilayer structures (vesicles) expressing a high membrane state of order with a distinct phase transition as typical for isolated LPS. However, in contrast to LPS, OML of T. denticola has a lower Tm near 22 degreesC and a lower cooperativity of the phase transition. The results suggest a different kind of permeation barrier that is built up by this particular OML of T. denticola, which is quite different from LPS normally essential for Gram-negative bacteria.
Subject(s)
Cell Membrane Permeability , Conotoxins , Lipopolysaccharides/chemistry , Membrane Lipids/chemistry , Neuropeptides/chemistry , Neuropeptides/metabolism , Treponema/chemistry , Electrophoresis, Polyacrylamide Gel , Fatty Acids/analysis , Mass Spectrometry , Membrane Fluidity , Molecular Weight , Spectroscopy, Fourier Transform Infrared , Teichoic Acids/chemistry , Treponema/ultrastructureABSTRACT
We have previously reported that bryostation 1 (Bryo 1) induces differentiation of chronic lymphocytic leukemia (CLL) in vitro to a hairy cell (HC) stage. This study tests the hypothesis that Bryo 1-differentiated CLL cells are more susceptible to 2-chlorodeoxyadenosine (2-CdA) than parent CLL cells. A recently established EBV-negative CLL line (WSU-CLL) from a patient resistant to chemotherapy including fludarabine was used to test this hypothesis. Both Bryo 1 (10-1000 nM) and 2-CdA (5.6-22.4 microM) exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. In vitro, the sequential exposure to Bryo 1 (100 nM for 72 h) followed by 2-CdA (11.2 microM) resulted in significantly higher rates of growth inhibition than either agent alone. Changes in immunophenotype, enzymes, lipids, proteins, and the DNA of WSU-CLL cells were studied before and after Bryo 1 treatment. Bryo 1 induced a positive tartrate-resistant acid phosphatase reaction and two important markers, CD11c and CD25, after 72 h of culture, confirming the differentiation of CLL to HC. The Fourier transformation infrared spectroscopic analysis showed that the amount of membrane lipids significantly increased in Bryo 1-treated cells compared to controls after 24 h, whereas the protein content, as well as the DNA content, decreased. This finding supports the change of CLL to HC. To evaluate the in vivo efficacy of Bryo 1 and 2-CdA, we used a xenograft model of CLL in WSU-CLL-bearing mice with severe combined immune deficiency. s.c. tumors were developed by injection of 10(7) WSU-CLL cells, and fragments were then transplanted into a new batch of severe combined immunodeficient mice. Bryo 1 and 2-CdA at the maximum tolerated doses (75 micrograms/kg i.p. and 30 mg/kg s.c., respectively) were administered to the mice at different combinations and schedules. The survival in days, the tumor growth inhibition ratio, the tumor growth delay, and the log10 kill of the mice treated with Bryo 1 followed by 2-CdA were significantly better than the control and other groups. We conclude that the sequential treatment with Bryo 1 followed by 2-CdA resulted in higher antitumor activity and improved animal survival.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Acid Phosphatase/metabolism , Aged , Animals , Apoptosis/drug effects , Bryostatins , Cell Division/drug effects , Cladribine/administration & dosage , DNA, Neoplasm/metabolism , Drug Administration Schedule , Drug Screening Assays, Antitumor , Humans , Lactones/administration & dosage , Leukemia, Hairy Cell/metabolism , Lipid Metabolism , Macrolides , Male , Mice , Mice, SCID , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Spectroscopy, Fourier Transform Infrared , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Precise regulation of cell organization results in well-differentiated tissue structures and continuous renewal of the oral epithelium maintaining a highly ordered tissue architecture. Here we demonstrate that FT-IR microspectroscopy, performed on sections of cancerous tissue biopsies, is capable of generating biochemical maps that show the distribution and any abnormal concentration of individual classes of biomolecules. Oral epithelia affected by cancer (squamous cell carcinoma) show many abnormal changes in morphology, of which the formation of keratin pearls is only one. Spectra from selected pearl areas demonstrate that these structures contain not only abnormal keratin concentrations but also seem to be stabilized by surrounding collagen fibers. Infrared image maps reveal that in the center of keratin pearls the concentration of protein (cytokeratins) is abnormally high, that DNA is absent and that the cell membrane fluidity is reduced. This suggests that cells are structurally destroyed and transformed into nuclei-free horny cells, simulating normal differentiation and epithelial growth. We also introduce a new analysis modality, two-dimensional (2D) tissue classification, and apply it to establish spectral similarities between different tissue structures. A total of 315 spectra, recorded for the original map, were analyzed by pattern recognition methods, classified and re-assembled into new maps based on their spectral similarities. The re-assembled maps clearly indicate significant tissue changes outside the pearls, suggesting early biochemical changes that accompany abnormal growth. Employing this 2D analysis modality in combination with infrared histopathology may be relevant to tumor diagnosis and prognosis.
Subject(s)
Keratins/analysis , Mouth Neoplasms/metabolism , Neoplasms, Squamous Cell/metabolism , Spectroscopy, Fourier Transform Infrared/methods , Cluster Analysis , Humans , Mouth Neoplasms/classification , Mouth Neoplasms/pathology , Neoplasms, Squamous Cell/classification , Neoplasms, Squamous Cell/pathologyABSTRACT
CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect.
Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Escherichia coli/enzymology , Nucleoside-Phosphate Kinase/metabolism , Phosphotransferases , Amino Acid Sequence , Hot Temperature , Molecular Sequence Data , Nucleotides/metabolism , Protein Binding , Protein Denaturation , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Spectrophotometry, InfraredABSTRACT
The drug resistance of leukemic cells from 21 patients with chronic lymphocytic leukemia (CLL) to the alkylating agent chlorambucil (CLB) and the nucleoside analog cladribine or 2-chlorodeoxyadenosine (CdA) was investigated by infrared spectroscopy. Drug sensitivities, determined in vitro with the tetrazolium dye (MTT) assay, were correlated with the infrared spectra of the CLL cells, applying linear discriminant analysis (LDA). The 63 spectra (three from each of the 21 samples), obtained before drug exposure, were successfully partitioned into drug-sensitive and drug-resistant groups; the LDA-based ex vivo prediction of the sensitivity to CdA or CLB was 85.7% and 80.3%, respectively. Similar changes in the composition/structure of DNA were observed between the spectra of the drug-sensitive and drug-resistant CLL cells for both CdA and CLB. However, CdA-resistant CLL cells could also be differentiated from CdA-sensitive CLL cells by spectral changes associated with membrane lipids; these differences were much less pronounced between CLB-resistant and CLB-sensitive CLL cells. We demonstrate here for the first time that infrared spectroscopy can be used as a new tool for predicting ex vivo drug response (sensitivity/resistance).
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Adult , Aged , Aged, 80 and over , Chlorambucil/administration & dosage , Cladribine/administration & dosage , DNA, Neoplasm/blood , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Humans , Lipids/blood , Lymphocytes/metabolism , Male , Middle Aged , Neoplasm Proteins/blood , Predictive Value of Tests , Prognosis , Spectroscopy, Fourier Transform Infrared , Tetrazolium Salts , Thiazoles , Tumor Cells, CulturedABSTRACT
A new quantitative method, based on Fourier transform infrared spectroscopy, was developed to evaluate the thiocyanate concentration in human saliva. Saliva samples were collected following a typical protocol and infrared spectra obtained from very small volumes (5 microl) deposited on a barium fluoride substrate. Exogenous potassium thiocyanate was used for calibration of the endogenous thiocyanate. This methodology does not require separation or extraction procedures. Human saliva spectra contain a characteristic marker band, due to thiocyanate, at 2058 cm-1. The integrated area of this band can be used for linear regression analysis and provides a good correlation between band area and thiocyanate concentration. Recovery of thiocyanate added to saliva was 100%. Centrifugation and dialysis experiments demonstrated that thiocyanate in saliva exists as a free or loosely bound ion. Saliva collected in the afternoon from 25 different subjects had a thiocyanate concentration of 0.83 +/- 0.42 (mean +/- SD) mmol/liter. In 4 subjects whose circadian pattern was investigated there was evidence of a higher thiocyanate concentration in saliva samples collected in the morning hours.
Subject(s)
Environmental Monitoring/methods , Saliva/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Thiocyanates/analysis , Cyanates/analysis , Humans , Reproducibility of ResultsABSTRACT
The peripheral mononuclear cells from 23 normal individuals and the purified B cells from 38 patients with chronic lymphocytic leukemia (CLL) were examined by Fourier transform infrared (FT-IR) spectroscopy. Differences were observed between the CLL and normal cells at the DNA, protein and lipid levels, with CLL cells having greater DNA and lower lipid contents than normal cells. In addition, the spectral character of the CLL and normal cells varied, demonstrating that there were also qualitative differences in the DNA and lipids. Statistical analysis, based on hierarchical clustering, separated normal from CLL cells completely and classified them into two subgroups for normal cells, while the CLL cells could be divided into three subgroups that were distinct from the normal cells. These differences were based on the lipid and DNA content and the overall spectral character of the cells.