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Study Objective: To investigate the ability of the cation exchange resin sodium polystyrene sulfonate to bind iron from ferrous sulfate solutions, along with the effects of pH on binding., Methods: We performed a series of in vitro experiments in which various concentrations of iron and sodium polystyrene sulfonate were combined and free ferrous iron was measured with the use of a colorimetric assay., Results: Sodium polystyrene sulfonate bound iron from ferrous sulfate solutions at pH 2 and pH 7. Slightly less binding of free ferrous iron was demonstrated in experiments performed at pH 7 than in those performed at pH 2. At pH 2, 98% of iron was bound, at pH 7, 95% of iron was bound., Conclusion: Sodium polystyrene sulfonate may be a useful therapy in acute iron poisoning once safety and efficacy are determined with the use of in vivo models.

The purpose of this study was to determine if the exchange resin sodium polystyrene sulfonate increases the clearance of lithium in a healthy volunteer. A single healthy volunteer received 900 mg lithium for each of three study phases. A sodium polystyrene sulfonate dose of either 0, 20, or 40 grams was administered one half hour after each lithium dose to assess the effect on lithium clearance. Multiple blood samples were obtained for serum lithium concentrations over a 36- to 48-hour time period. Pharmacokinetic parameter estimates were calculated and compared. Sodium polystyrene sulfonate was found to increase the clearance of lithium in a single volunteer. Sodium polystyrene sulfonate may be useful in the treatment of lithium intoxication by increasing the clearance of lithium.

Sodium polystyrene sulfonate, a cation exchange resin, should be useful in the treatment of lithium overdosage. This in vitro study was conducted to assess the ability of sodium polystyrene sulfonate to bind lithium, effects of pH on binding, binding efficacy in comparison to charcoal, and affinity for lithium versus potassium. Stock solutions of lithium were added to fixed amounts of sodium polystyrene sulfonate and charcoal. Lithium and potassium concentrations in supernatant were measured by flame photometry. Increasing concentrations of sodium polystyrene sulfonate bound more lithium. Changes in pH had little effect on lithium binding. Lithium is bound to sodium polystyrene sulfonate more readily than to charcoal. Potassium is preferentially bound to sodium polystyrene sulfonate over lithium. Sodium polystyrene sulfonate may provide a useful therapeutic modality in the treatment of lithium overdosage.

This report describes the development of an HPLC-UV method for studies of glycoamines and glycoamine-like compounds in normal human serum and osteosarcoma patients serum as potential biological markers of cancer. The glycoamines, a newly recognized class of endogenous, low-molecular-mass biopolymers, are conjugates of amino acids and sugar units, containing 5 to 29 amino acid and 1 to 17 sugar units. After ultrafiltration of serum samples, reversed-phase HPLC separation with diode-array detection was used to obtain standard profiles of serum ultrafiltrates below M(r) 10,000 in healthy subjects. These highly reproducible profiles utilized two-dimensional peak identification and were used to develop a statistical profile of the major glycoamine peaks in normal serum. This newly developed analytical method was subsequently used to address a key question: whether or not there is a single tumor-specific glycoamine or a family of tumor-specific glycoamines in cancer patient serum. Preliminary results suggest that this method can separate and detect glycoamines and glycoamine-like compounds in various types of cancer patients serum with a high degree of reproducibility on the basis of comparative two-dimensional identification of natural compounds and a panel of synthetic glycoamine analogs. Moreover, the method is useful for following the relative changes in the amount of a given glycoamine over an extended clinical time course. Initial results suggest that a glycoamine or glycoamine-like compound, GA-4.63, may have clinical utility in human osteosarcoma studies.

To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA. The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated. We have compared the ability of the mutant glycine tRNA1(UCC) and glycine tRNA1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons. The results showed that glycine tRNA1(ACC) unlike glycine tRNA1(UCC) did not fully discriminate between the glycine codons. These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons. In the present paper we give a detailed description of this new in vitro system.

Methylation was investigated in compositional fractions of nuclear DNA preparations (50-100 kb in size) from five plants (onion, maize, rye, pea and tobacco), and was found to increase from GC-poor to GC-rich fractions. This methylation gradient showed different patterns in different plants and appears, therefore, to represent a novel, characteristic genome feature which concerns the noncoding, intergenic sequences that make up the bulk of the plant genomes investigated and mainly consist of repetitive sequences. The structural and functional implications of these results are discussed.

Methylated purines and pyrimidines derived from the degradation of transfer ribonucleic acid have been shown to be excreted in abnormal amounts in the urine of patients with cancer. Recent technology developed by Gehrke and Kuo has allowed the separation and quantification of modified nucleosides in serum using reversed-phase high-performance liquid chromatography with diode-array measurement. Serum levels of ten modified nucleosides were measured in 37 normal healthy adults to establish normal values and to correlate activity with age and sex. In addition, serum levels of patients with several malignancies were measured to determine activity in these diseases. Levels of modified nucleosides in normal individuals were consistently reproducible and showed no significant variation among males versus females or with age. Patients with malignant diseases showed consistent elevations and these were highest in patients with more advanced disease. The evidence of no significant differences in the mean levels of modified nucleosides in serum with age or sex in normal adults and elevations in patients with malignancies demonstrate the potential value of modified nucleosides as cancer biomarkers.

We used a recently devised HPLC method to quantify eight modified nucleosides, an emerging group of tumor markers, in human serum and then calculated their reference intervals in a healthy population from Italy and the United States. We used the statistical procedure of element analysis, which reveals the effects of chosen variables (in this case, nationality, sex, and age) on an analyte (here, modified nucleosides). Using element analysis, we calculated the exact weight of each variable on the reference values. We found that nationality has the greatest effect on the serum concentrations of all the modified nucleosides apart from pseudouridine, whereas sex significantly influences only the concentrations of 4-pyridone-3-carboxamide-N1-ribofuranoside, 1-methylinosine and N2,N2-dimethylguanosine; age affects only N2,N2-dimethylguanosine. Thus, the reference intervals of all the nucleosides except pseudouridine were calculated separately for Italians and Americans, and the reference values for 4-pyridone-3-carboxamide-N1-ribofuranoside, 1-methylinosine, and N2,N2-dimethylguanosine were calculated separately for men and women. Our data form the baseline for study of variations in serum concentrations of modified nucleosides in various pathophysiological conditions.

The unknown modified nucleotide G*, isolated from both Schizosaccharomyces pombe and Torulopsis utilis initiator tRNAs(Met), has been identified as an O-ribosyl-(1"----2')-guanosine-5"-phosphate, called Gr(p), by means of HPLC, UV-absorption, mass spectrometry and periodate oxidation procedures. By comparison with the previously published structure of Ar(p) isolated from Saccharomyces cerevisiae initiator tRNA(Met), the (1"----2')-glycosidic bond in Gr(p) has been postulated to have a beta-spatial conformation. The modified nucleotide Gr(p) is located at position 64 in the tRNA(Met) molecules, i.e. at the same position as Ar(p). Since we have also characterized Gr(p) in Candida albicans initiator tRNA(Met), the phosphoribosylation of purine 64 can be considered as a constant nucleotide modification in the cytoplasmic initiator tRNAs(Met) of all yeast species so far sequenced. Precise evidence for the presence of Gr(p) in initiator tRNAs(Met) of several plants is also reported.

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65.

The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry. The structure was established as 5-carbamoylmethyluridine (ncm5U). In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon. In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same 'body tRNA' with one posttranscriptional exception at position 32, where pseudouridine is associated with ncm5U (position 34) in tRNA(Pro) (U*GG) and 2'-O-methylpseudouridine is associated with inosine (position 34) in tRNA(Pro) (IGG).

The modified nucleoside (U*) present in the wobble position of Saccharomyces cerevisiae mitochondrial tRNA(Leu) and tRNA(Trp) was isolated by thin-layer chromatography and HPLC. Its chromatographic, UV spectral, and mass spectrometric properties were shown to be identical with those of 5- [[(carboxymethyl)amino]methyl]uridine (cmnm5U). This nucleoside found in yeast mitochondrial tRNAs reading two-codon families ending in a purine permits the selective recognition of A and G in the third codon position.

The monitoring utility of serial patterns of a single marker (carcinoembryonic antigen) level and multiple marker (three carbohydrates-fucose, mannose and galactose) levels in patients with small cell carcinoma of the lung was investigated using a quantitative approach. A serial multiple regression (SMR) model was formulated to assess the disease response following the first to the tenth course of therapy and resulted in the multiple correlations ranging from 0.183 (NS)-0.706 (P less than 0.005) for CEA, and 0.502 (P less than 0.250)-0.760 (P less than 0.025) for three carbohydrates, respectively. The appraisal of these markers utilizing the SMR model points out that: (1) the serial levels of CEA greater than 5.0 ng/ml are significantly correlated with the disease course whereas the serial levels less than 5.0 ng/ml reflect the trend of variation in the disease course, but with less accuracy; and (2) the levels of three carbohydrates, any one elevated before and during therapy, are significantly correlated with the disease course.

Clinical correlates with urinary excretion of polyamines were evaluated for 29 newly diagnosed and 35 previously treated patients with small cell carcinoma of the lung (SCC). The frequencies of pretreatment abnormalities were 12 (41%) for putrescine, 18 (62%) for spermidine, and 20 (69%) for spermine. In assessing disease parameters, the combined use of the abnormalities of spermidine and spermine as a discriminant was more effective than that of all three polyamines; it correlated significantly with extent of limited and extensive disease (P less than 0.001), and also resulted in significant separation of survival curves, the median survival of 11 months for both elevated compared to 19 months for neither or only one elevated (P = 0.062). No significant difference was seen in the abnormalities between no metastasis and one metastasis, whereas the frequencies of the abnormalities was highly increased in two or more metastases. The distribution of polyamines determined at regular treatment intervals showed distinctively more elevated patterns in progressive disease than in stable disease or partial and complete responses (P less than 0.01). In order to evaluate therapeutic effects on the relationship between polyamine excretion and tumor regression, correlations between urinary putrescine and spermidine were determined. The values of the ratio of spermidine to putrescine were significantly smaller in responders than in nonresponders (P less than 0.01); and these may be related to smaller tumor mass and higher tumor proliferative activity in responders, and larger tumor mass and lower tumor proliferative activity in nonresponders.

Enzymatic hydrolysis and base analysis by high performance liquid chromatography showed that mouse satellite DNA had 30-50% less 5-methylcytosine in sperm than in somatic tissue (1.59 mols % vs 2.40-3.11 mols %). Maxam-Gilbert sequencing and analysis of the intensity of the cytosine bands indicated that the level of methylation of the eight CpGs of the consensus sequence in sperm satellite DNA ranged from 0 to about 50%, considerably lower than the levels reported in somatic tissues. The Mn1I site containing one of these CpGs was cut much more extensively in satellite DNA from sperm than from liver, confirming the undermethylation of this site in sperm DNA.

Urinary levels of the minor nucleosides, pseudouridine (psi),N2, N2-dimethylguanosine (m22G), and 1-methylinosine (m1I), were investigated in patients with breast carcinoma. Elevated levels of psi were observed in 27/131 (20.6%) patients with metastatic disease, 1/14 (7.1%) preoperative patients, and 1/28 (3.6%) postoperative N+ patients. Elevated levels of m22G and M1I were observed, respectively, in 46/131 (35.1%) and 274131 (20.6%) patients with metastatic disease, 3/14 (21.4%) and 3/14 preoperative patients, and 6/28 (21.4%) and 2/28 (7.1%) postoperative N+ patients. There was no correlation between nucleoside levels and involvement of specific organ sites with metastatic disease, nor with chemotherapy response rate or time to treatment failure. During the treatment of metastatic disease there was a tendency for elevated pretherapy psi levels to decrease with attainment of a response and, if the levels subsequently rose to be associated with treatment failure. However, increasing levels of m22G and 71I occurred with both response and disease progression. These results suggest that routine measurement of the level of the urinary nucleosides would be of limited value for following the disease course in patients with breast cancer.

Cancer patients and tumor-bearing animals excrete high levels of modified purines and pyrimidines some of which, e.g., N2,N2-dimethylguanosine, can originate only from transfer RNA (tRNA). Until recently, it could not be ascertained whether the high level of excretion of such compounds is due to cell death or specific tRNA turnover. However, an approach to this problem became feasible, with beta-aminoisobutyric acid as a probe. This compound is a terminal degradation product of thymine which is present in both DNA and tRNA. Since the pathway of synthesis of thymine is different in the two macromolecules, it and its end product, beta-aminoisobutyric acid can be differentially labeled with [14C]formate and [3H3]methylmethionine as precursors. Therefore the ratio of the two labels in the excreted beta-aminoisobutyric acid is a measure of the macromolecular origin of the degradation product. We have found from such analysis that tRNA's are not homogeneous in their turnover rate. There is a subpopulation that turns over much faster than the rest. The turnover rate of a subpopulation of tRNA's in tumor tissue exceeds the turnover rate of tRNA's in normal tissue. Such rapid degradation of tRNA's must be the source of the massive excretion of modified nucleosides by cancer patients which can be 10-fold higher than in normal subjects.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.

The sulfur-containing amino acids cystine and methionine play important roles in animal, especially avian, nutrition. Because these sulfur-containing amino acids are destroyed to varying extents by 6N HCl hydrolysis, oxidation and hydrolysis of cystine to cysteic acid and methionine to methionine sulfone have been widely used for determination of cystine and methionine. Lysine is considered the next limiting amino acid after the sulfur amino acids in poultry nutrition; therefore, determination of the amino acid content of rations focuses first on these 3 amino acids. The objective of this investigation was to establish whether lysine and other amino acids could be accurately determined in proteinaceous materials which had undergone performic acid oxidation. To perform this evaluation, lysine was determined in a variety of protein-containing materials both with and without performic acid oxidation. Performic acid oxidation followed by 6N HCl hydrolysis at 145 degrees C for 4 h allows accurate measurement of 3 amino acids especially important to poultry nutrition, cystine, methionine, and lysine, in a single preoxidized hydrolysate; this method can be extended to another 9 protein amino acids.

The level of DNA methylation in three mouse-human cell lines that retained different human chromosomes and in the parental mouse and human lines has been determined by high-pressure liquid chromatography (HPLC). The level of methylation is similar in the hybrid and parental cells, indicating that interspecific somatic cell hybridization followed by preferential chromosome segregation can occur without an increase in overall DNA methylation.

Serum fucose-protein ratio was evaluated as a potential biologic marker for patients with metastatic breast cancer. By analysis of the same blood samples, comparisons were made with carcinoembryonic antigen (CEA) and human chorionic gonadotrophin (hCG). For 150 patients with metastatic breast cancer, 85% had a value for serum-fucose protein ratio above the normal range in comparison to 75% for CEA and 40% for hCG. Serum fucose-protein ratio was exclusively increased in 12% of the patients, CEA in 4% and hCG in 2%. Both serum-fucose protein ratio and CEA were elevated in 39% of the patients, and together, either in combination of alone, were increased in 93% of the patients. Raised values for serum fucose-protein ratio as well as for CEA decreased with change in disease status from pretreatment to response for patients with measurable disease parameters and increased correspondingly with overt disease progression. Preliminary data indicate both serum fuxose-protein ratio and CEA frequently become elevated when patients progress from a disease free interval after surgery to recurrence.

Five minor base ribonucleosides, primarily degradation products of transfer ribonucleic acid (tRNA), were evaluated as potential biological markers for patients with small cell carcinoma of the lung. The urinary concentration for pseudouridine, 1-methyladenosine, 1-methylinosine, N2-methylguanosine, and N2,N2-dimethylguanosine was determined by means of reversed-phase high performance liquid chromatography and quantitatively expressed as a function of creatinine excretion. Comparisons were made with carcinoembryonic antigen (CEA) plasma levels. The total frequency of elevated values for the five nucleosides in pretreatment urine samples was directly related to stage of disease with 24/60 (40%) determinations increased in 12 patients with limited disease and 69/85 (81%) in 17 patients with extensive disease. For these same patients, CEA levels were elevated respectively in 2/11 (18%) of the former and 9/17 (53%) of the latter group. The frequency and degree of elevation of the nucleoside/creatinine ratios in pretreatment samples from patients with extensive disease was correlated directly with increasing number of metastatic sites. Of the five nucleosides, the mean number elevated was two for limited disease, 3-4 for extensive disease with one metastatic site, 4 for two or three, and 5 for four or more sites of metastases. Based on a summation of pretreatment nucleoside/creatinine ratios, a discriminant for survival was derived giving curves separating patients (P = 0.086) similar to the discriminant based on stage of disease. Although discordant results were noted, an overall correlation of 75% agreement with clinical assessment was estimated in response categories when monitoring changes associated with therapy.

The lunar samples from Apollo flights 11 through 17 provided the students of chemical evolution with an opportunity of examining extraterrestrial materials for evidence of early prebiological chemistry in the solar system. Our search was directed to water-extractable compounds with emphasis on amino acids. Gas chromatography, ion-exchange chromatography and gas chromatography combined with mass spectrometry were used for the analysis. It is our conclusion that amino acids are not present in the lunar regolith above the background levels of our investigations.

[We have measured four urinary nucleosides (dimethylguanosine, 1-methylinosine, pseudouridine and beta-aminoisobutyric acid)in patients with benign breast disease and patients with early and advanced breast cancer in order to assess their value as tumour-index-substances. We compared the results with other biochemical indices of breast cancer and sought and correlations between these indices. The results indicate that few abnormalities occurred in patients without overt metastases and these did not predict early relapse. In those with metastatic disease, dimethylguanosine excretion was most frequently elevated. Correlations were observed between some of the nucleosides and lysozyme and alpha1-antirypsin.

A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed. We developed a simple sample cleanup in which the samples were thawed, neutralized with KOH, immediately derivatized, extracted into ethyl acetate (EtOAc) and then chromatographed by HPLC. The derivatives were stable in EtOAc for more then 24 h. Interfering amino acids were removed from the EtOAc by partitioning twice with Na2HPO4 buffer (pH 10.0). Complete separation was achieved in ca. 60--90 min on a muBondapak phenyl column using a stepwise gradient of acetonitrile and/or methanol-phosphate buffer (pH 5.1). A variable wavelength fluorometer with a 5-microliter flow-cell was used (excitation 340 nm; emission 480 nm). Linearity ranged from 200 pg to 50 ng onto the column. Precision (R.S.D.) for retention times was 1% and for derivatization and injection 2.5%. Recoveries of the seven biogenic amines from plasma spiked with 25 ng/ml averaged 70%, with a relative standard deviation of 6%. Separation studies were also done using a muBondapak C18 column. The effects of various eluents are presented. Gas-liquid chromatography was also investigated but lacked the sensitivity achieved by HPLC. The HPLC method is used routinely for the determination of biogenic amines in plasma from pigs with malignant hyperthemia and thermally stressed bovine. Significant differences in levels of biogenic amines were noted between stressed and non-stressed animals. Data on rat brain tissue samples were compared with the trihydroxyindole method and canine heart tissue was analyzed for ventricular norepinephrine and dopamine. Application of the method to urine from normal persons and a patient with a brain tumor has been demonstrated.

The heat-induced conversion of 5-methylcytosine (m5C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37 degrees C and pH 7.4 were approximately 9.5 X 10(-10) and 2.1 X 10(-10) sec-1, respectively. N4-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N4-methylcytosine rather than m5C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m5C in their DNA.

The massive amounts of beta-aminoisobutyric acid (beta-AIBA) in the urine of Burkitt's lymphoma patients were measured along with other alpha-amino acids and beta-alanine present in normal and decreased levels. The ratios of the amount of beta-AIBA to beta-alanine, in mumoles/kg urine collected in 24 hours, were elevated for all patients. The degree of elevation of beta-AIBA excretion and the ratio of the two beta-amino acids appeared to be related to the amount of tumor mass present. These analyses may have possible value in monitoring patients with Burkitt's lymphoma during their disease course.

This presentation describes amino acid analysis with the gas chromatographic method and experimental conditions using the N-trifluoroacetyl n-butyl ester derivatives; the study we describe here was undertaken to compare gas chromatographic (GC) and ion-exchange chromatographic (IEC) analyses of amino acids in hydrolysates of 9 diverse sample types to gain insight into effects of these 2 chromatographic methods of analysis on variation in amino acid results. Our study showed that values for samples prepared by 2 separate laboratories using the same procedure were generally in good agreement when all of the hydrolysates were analyzed by a single laboratory using a single method of analysis. To compare results from gas chromatography with those from ion-exchange chromatography analyses were performed by 2 different laboratories on the same hydrolysates and on different hydrolysates prepared by the same method by both laboratories. The data demonstrate that GC and IEC can be expected to yield essentially identical results when applied to the same hydrolysate. Agreement is so close that interlaboratory differences in hydrolysate preparation of the same sample contribute as much to variation in amino acid results as does the method of analysis, a fact which should be noted in planning collaborative studies.

Under the conditions of disynchronization by the manipulation of both the alternation of light and darkness and the availability and unavailability of food, circadian rhythms characterize the excretion of several amino acids by inbred LOU rats bearing an immunocytoma. Large amplitude rhythms can be demonstrated for urinary beta-aminoisobutyric acid, beta-alanine, phenylalanine and tyrosine. Under the same conditions of disynchronization, control animals excrete the same compounds also with a marked circadian variation but at an invariably lower average rate. These excretory rhythms, along with those demonstrated earlier for polyamines and light-chains, are of interest as potential markers for the chronotherapy of cancer.

To compare the fatty acid distribution and cholesterol composition in trained and untrained isolated skeletal muscle membranes, a needle biopsy was performed on the vastus lateralis of the 10 distance runners and 10 sedentary men. The muscle sample was homogenized and centrifuged at 100,000 X g; the resulting pellet was analyzed by gas-liquid chromatography for individual fatty acids and cholesterol. The percentage of palmitic acid was significantly lower in the trained muscle tissue. Samples from the distance runners also tended to have a more frequent appearance of linolenic and eicosatrienoic acids, longer fatty acid hydrocarbon chains, and lower cholesterol concentration. It was concluded that trained muscles have an increased membrane fluidity which could beneficially affect the activity of membrane-bound enzymes and active transport. Longer chain length in the membrane lipids may be a means of producing an inner membrane cohesiveness in muscles of trained individuals.

A major glutamine tRNA from rat liver was purified. Post-labeling techniques showed its nucleotide sequence to be: pG-G-U-U-C-C-A-U-m(1)G-G-U-G-psi-A-A-D-Gm-G-D-D-A-G-C-A-C-U-C-U-G-G-A-Cm-U-C-U-G-A-A-psi-C-C-A-G-C-G-A-U-m(5)C-m(5)C-G-A-G-psi-psi-C-A-m(1)A-A-U-C-U-C-G-G-U-G-G-A-A-C-C-U-C-C-A(OH).Images

The aims of this investigation were to study the completeness of silylation of nucleosides with three different reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA), bis(trimethylsilyl)acetamide (BSA) and trimethylsilylimidazole (TMSI), and to investigate the effect of different solvents (acetonitrile, pyridine, dimethylformamide, chloroform, methylene chloride, hexane, benzene, and toluene) on quantitation of derivatization. Closed-tube silylations of the nucleosides were performed with BSTFA, BSA, and TMSI, and for the most complete silylation, the optimal time, temperature, and molar excess of reagent were: for BSTFA, 150 degrees-15 min and 225 molar excess; for TMSI, 60 degrees-3 h and 1000 molar excess; and for BSA, 120 degrees-2 h and 250 molar excess. Also, silylations of seven major and minor nucleosides were carried out using a 1000 molar excess of BSTFA, BSA, and TMSI at 25 degrees with 5 min sonication, and at optimal silylation conditions as described above for the three reagents. The silylating strengths were determined by the increase in RWR (= weight response of nucleoside/weight response of pyrene) values, and are summarized for the amino group containing nucleosides silylated at room temperature as BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions as BSTFA greater than BSA greater than TMSI. The efficiency of silylation for the hydroxyl group-containing nucleosides silylated at room temperature was BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions BSTFA greater than TMSI = BSA...

A minor modification in the automated analytical system of the official AOAC semiautomated method for determining crude proteins results in an automated method for determining urea and ammoniacal nitrogen in animal feeds and their ingredients. Urease enzyme which has high activity, yields a clear solution in water, has low ammonia impurity, and is inexpensive is used in the automated method. Weights from 1 to 2.5 g feed sample are dissolved in water, and sample solutions are analyzed at the rate of 40 samples/h. Five AAFCO feed check samples were analyzed repeatedly by the automated method, and results were compared with the grand averages from the check sample reports. The official AOAC manual urease method was used by AAFCO participants. Average recovery of urea and ammoniacal nitrogen was 100.6% by the automated method relative to the AAFCO reported averages. The range of recoveries as 98.5-102.7%. The non-protein nitrogen (NPN) concentrations, expressed as protein equivalent, ranged from 3.40 to 63.04% protein on these samples. The average relative standard deviation for the automated analyses was 0.77%, compared with 1.54% for the manual method. This method is an important adjunct to laboratories using or considering use of the semiautomated method for crude protein and needing further information on NPN.

Urinary polyamine levels were evaluated in patients with breast carcinoma. The individual levels of putrescine, spermidine, spermine, and cadaverine, and the product/precursor levels of putrescine, spermidine, and spermine were analyzed. Elevations of one or more individual polyamines or of the ratios were found in 50% of patients with metastatic disease, 38.5% of preoperative patients, and 35.7% of 5--24 week postoperative N + patients. Sequential sampling of patients with metastatic disease suggested that changes in elevated polyamine levels tend to reflect the clinical course of the disease, especially for the association of treatment failure with rising elevated values. The presence of one or more elevated parameters prior to treatment of metastatic disease tended to be associated with a higher response rate (85.7 vs. 68.4%) than all normal levels. Five of nine patients who recurred postoperatively had preceding postoperative polyamine elevations. In addition, there was a trend for a shorter disease-free time among patients with one of more elevated polyamine parameters between 5--24 weeks postoperatively than among patients with normal parameters. These data suggest that measurement of urinary polyamine levels, including calculation of the product/precursor levels, may be a useful clinical adjunct in the management of patients with breast carcinoma.

The aims of this investigation were to establish the optimum reaction conditions for silylation of nucleotides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and to investigate the chromatographic properties of the following nucleosides: adenosine, guanosine, cytidine, thymine, inosine, xanthosine, and uridine. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150, and 175 degrees for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150 degrees and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500, and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1-7 days was investigated. Quantitative gas-liquid chromatography of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (w/w) SE-30 on Supelcoport, different loadings (%, w/w) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (w/w) OV-11 or 3% (w/w) OV-17 or 4% (w/w) Dexsil-300 on Supelcoport. A complete separation of the six ribonucleosides including guanosine and cytidine was obtained with 1 m x 4 mm I.D. glass columns of 4% (w/w) OV-11 and 3% (w/w) OV-17 on 100-120 mesh Supelcoport. It was observed that with 2'-deoxycytidine, one obtains the TMS cytosine peak (retention temperature 150 degrees) plus another peak with a retention temperature of 120 degrees, under all derivatization conditions with BSTFA and bis(trimethylsilyl)-acetamide. Similar formation of bases from other 2'-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5 to 10 ng injected with a signal-to-noise ratio of 3. The relative standard deviations for all nucleosides and deoxynucleosides ranged from 1.2 to 4.8% on different methylsiloxanes on Supelcoport columns.

By means of a sensitive and specific method utilizing gas-liquid chromatography, the excretion levels for three nucleosides, degradation minor base products of ribonucleic acid, primarily transfer ribonucleic acid, were determined in 24-hour urine specimens from over 200 patients with solid tumor malignancies. These nucleosides were N2,N2-dimethylguanosine, l-methylinosine, and pseudouridine. When compared to normal control values, elevated levels of these compounds were found for patients in each of several tumor types studied. Increases in pseudouridine excretion suggest increased tumor transfer ribonucleic acid turnover; in addition, for the methylated nucleosides, higher than normal values may reflect enhanced transfer ribonucleic acid methylase activity of the neoplastic cells.

Levels of putrescrine, spermidine, and spermine in urine were determined by means of a sensitive ion-exchange chromatographic method in patients with advanced solid tumor malignancies, in patients with diseases other than cancer, and in normal control subjects. Elevation above 2 SDS of the normal mean were found in varying number of patients in each tumor category. For those malignancies studied that involved more than 20 patients, the greatest incidences of increased excretion were 66% for spermine in patients with colon carcinoma and 50% for putrescine and spermidine in patients with bronchogenic carcinoma. The highest levels and greatest frequency of elevated polyamine levels were found in patients with Burkitt's lymphoma, and changes in clinical tumor status associated with treatment appeared to correlate well with polyamine levels in this disease. Abnormal amounts of polyamines were also excreted by some patients with diseases other than cancer, indicating that increased polyamine excretion is not restricted or specific to the neoplastic state. It was also found that the levels of polyamines were apparently not affected by the intake of meat or the diet eaten, and remained in a rather narrow excretion range for any one individual at different time intervals. This study was carried out as part of a program to determine and evaluate biologic materials present in body fluids that may be used to follow and evaluate response or progression of neoplastic disease in patients during treatment regimens. The results suggest that abnormal urinary polyamine levels may be characteristic of neoplastic growth for some patients with malignant disease. Further studies are necessary to determine if these compounds may be helpful in assessing disease status for patients with such solid tumor malignancies as colon and bronchogenic carcinoma although their potential as useful "biologic markers" appears less promising than originally anticipated.

Seven breakdown products of tRNA were quantitated by high pressure liquid chromatography in urine and were related to the creatinine content. In the urine of 26 of 27 patients with 13 different malignancies, there was an elevation of one or more of these "markers." The levels of excretion vary approximately with the stage of the disease.

A method is described for the analysis of fecal neutral steriods with a dual-column gas-liquid chromatography (GLC) system. After saponification of the fecal slurry, the neutral steroids were extracted with hexane. The GLC separation of the compounds and quantitation were achieved by simultaneous injection of the derivatized and derivatized aliquots of the extract onto dual colmuns under identical conditions. The neutral steroids of interest were than identified by matching the retention times with those of known standards, and identification was confirmed by use of an interfaced GLC high-resolution mass spectrometry system. The detection limit was 0.003 mg of steroid/g of fecal slurry. The pricision of the method is illustrated by a relative standard diviation of 2-10% and a recovery of neutral steroids from 73-96%. The method was applied to the determination of fecal neutral steroids in a "High protein diet in colon cancer study". A considerably larger level of coprostanone than of coprostanol was observed. Data on neutral steroids in fecal samples from subjects on different diets are the subject of a separate publication.

In order to examine the relationship between fatty acid distribution of skeletal muscle membranes and age, a needle biopsy was performed on the vastus lateralis muscle of 20 healthy, non-obese males, ranging in age from 22 to 73 years. The muscle sample was homogenized, centrifuged at 100,000 x g, and the resulting pellet was saponified and acidified. The fatty acids and cholesterol were removed by a single hexane extraction and analyzed by gas--liquid chromatography with flame ionization detection. All subjects regardless of age had no consistent differences in the fatty acid profiles and cholesterol composition in the tissue. Correlation coefficients indicated no significant relationship between the age of the individual and any of the analyzed lipids. The results of this study indicated that aging may not be reflected by gross changes in the composition of structural lipids in the cell.

Malignant hyperthermia (MH) is a genetic disease of man, swine, dogs, cats, and horses. The syndrome is normally triggered by inhalational anesthetics or the administration of depolarizing muscle relaxants such as succinylcholine or various environmental stress factors. We have used the MH-susceptible pig as an animal model to study the hormonal changes developing during this highly lethal syndrome. High-performance liquid chromatography with electrochemical detection was used for the quantitation of the plasma levels of norepinephrine and epinephrine during MH. This research presents evidence that the rapid release of massive quantities of norepinephrine (up to 108 ng/ml) into the blood stream occurs simultaneously with the initiation of tachycardia which is the herald signal of the onset of MH. Norepinephrine levels exceed epinephrine by a 4:1 ratio early in the syndrome. Even pigs with MH which do not develop the muscle rigor phase have high levels of circulating norepinephrine. Tachycardia, pulmonary hypertension, increased venous oxygen desaturation, and increasing core temperature develop as the syndrome progresses.

5-methylcytosine residues in DNA underwent deamination at high temperatures. Furthermore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [14C]-5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [14C] 5-methylcytosine residues. Upon incubation at 95 degrees C in a physiological buffer or at 60 degrees C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those on cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.