Biomedical subjects

B W Patterson

Publications and source records attributed to B W Patterson.

At least 19 recordsLinked to original sources

PubMed1992

VLDL apolipoprotein B-100, a potential indicator of the isotopic labeling of the hepatic protein synthetic precursor pool in humans: studies with multiple stable isotopically labeled amino acids.

Four adult men received a 48-h constant intravenous infusion of [2H4]lysine, [2H3]leucine, L-[ring-13C6]phenylalanine, and L-[1,2,3,-13C3]alanine. Subjects ingested hourly meals for two 12-h periods, separated to two 12-h fasting periods. The isotopic enrichments of free amino acids in venous plasma and in VLDL apolipoprotein B-100 (apoB)-bound amino acids, plasma alpha-keto isocaproic acid (alpha-KIC) and plasma pyruvic acid (PYR) were measured by negative chemical ionization gas chromatography-mass spectrometry. By 7 h of infusion, all four amino acids achieved an equilibrium isotopic enrichment (EIE) in plasma and in apoB. In the fed state, the EIE of the amino acids in apoB was lower than that in plasma free amino acids. The ratio EIE-apoB:EIE-plasma differed significantly among amino acids in the fed state (alanine 0.30; lysine 0.64; leucine 0.70; phenylalanine 0.81). In the postabsorptive state, the EIE-apoB:EIE-plasma ratio rose significantly compared with the fed state (alanine 0.38; lysine 0.73; leucine 0.94; phenylalanine 1.05). Plasma PYR and apoB-alanine were in isotopic equilibrium irrespective of nutritional state. The EIE-apoB-leucine:EIE-plasma-alpha-KIC ratio rose from 0.75 in the fed state to near 1 in the postabsorptive state. We conclude that the contribution of systemic amino acids to apoB-100 synthesis is sensitive to nutritional state, and that systemic essential amino acids seem to be preferentially incorporated into apoB.

Adult↗

PubMed1992

Neonatal genetically lean and obese pigs respond differently to dietary cholesterol.

The impact of cholesterol exposure in early life on later cholesterol metabolism is not clearly understood. Sixteen newborn genetically lean and obese pigs were fed 0 or 5.0 g cholesterol/kg diet (0 or 0.5%) (liquid diets for 12 d, dry diets thereafter) for 33 d, after which they were all fed 10.0 g cholesterol/kg diet (1.0%) for 23 d. All animals were killed on d 56 and whole-body protein, fat and water were determined on the ground carcass. Dietary cholesterol had no consistent effect on growth rates or body composition. Mean fat content of lean pigs was 15.1% compared with 22.7% for obese pigs; corresponding values were 14.8 and 14.4% for protein and 65.5 and 58.3% for water. Concentrations of plasma total cholesterol, HDL cholesterol and apolipoproteins B and A-1 were increased by 0.5% dietary cholesterol in obese but not in lean piglets, although dietary cholesterol caused HDL and LDL size distribution profiles to shift toward larger-sized components in both strains. Plasma total cholesterol and apolipoprotein B concentrations rose two- to eightfold in all groups after the 1% cholesterol diet was consumed; these changes were accompanied by shifts in LDL and HDL size distribution profiles towards larger-sized components. With 1.0% cholesterol in the diet of all groups, HDL cholesterol concentration increased by approximately 50% in both groups of lean pigs and in obese pigs previously fed cholesterol, but did not increase further in obese pigs previously fed 0.5% cholesterol. The magnitude of the hypercholesterolemic response in lean pigs was blunted by previous exposure to 0.5% dietary cholesterol, but the response was accentuated in obese animals that had been previously exposed to 0.5% dietary cholesterol. These data provide evidence that genetic differences between obese and lean pigs affect their serum lipoprotein responses to high cholesterol intake.

Animals↗

PubMed1991

Isotopic determination of organic keto acid pentafluorobenzyl esters in biological fluids by negative chemical ionization gas chromatography/mass spectrometry.

A rapid, single-step procedure for the extraction and derivatization of organic alpha-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto acids because it does not introduce any additional isotopic complexity into the target molecule. Normal human plasma 4-methyl-2-oxopentanoic acid levels were 34.9 +/- 5.3 mumol.L-1 and could be determined with 1.1% precision by isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanoic acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.

Fluorobenzenes↗

PubMed1991

Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins.

Procedures are presented for the separation and determination of the isotopic enrichment of multiple human apolipoproteins labeled in vivo with a stable isotope amino acid. The isotopic enrichments of plasma lysine and plasma apolipoproteins were monitored for 16 days after a single intravenous dose of [4,4,5,5-2H4]lysine (5 mg/kg body weight). The use of a multiply deuterated amino acid enabled the measurement of isotopic enrichments above background over the entire 16-day time course in all proteins. Individual apolipoproteins were separated on a specially designed gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis system cast in a conventional slab gel apparatus which resolved apoB-100, apoE, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III-1, and apoC-III-2 on a single gel. After staining with Coomassie blue, proteins bands (containing 5 to 30 micrograms of individual apolipoprotein) were excised from the gel. Amino acids were recovered from hydrolyzed gel slices, derivatized, and analyzed by gas chromatography-mass spectrometry for determination of lysine isotopic enrichments. The utility of the method is demonstrated using examples of apolipoproteins B-100, A-I, A-II, C-I, C-II, and C-III from either total plasma d less than 1.21 g/ml lipoproteins or selected lipoprotein subfractions. Lysine isotopic enrichments of proteins were generally determined with a precision of better than 5%. The isotopic enrichment profiles were consistent with literature reports of apolipoprotein metabolic kinetics based on the use of radioiodinated apolipoproteins. The procedures outlined can be used to separate and measure the isotopic enrichment of virtually any apolipoprotein from any chosen lipoprotein fraction. Thus, these procedures should find wide application in the study of apolipoprotein metabolic kinetics.

Adult↗

PubMed1989

The relationship between neuropsychological and late component evoked potential measures in chronic alcoholics.

The relationships between event-related potential (ERP) measures and neuropsychological measures were investigated in a group of 39 male alcoholics and 22 age-matched male controls. Late component ERP measures such as N1, Nd, and P3 components and neuropsychological measures of perceptual-motor function, semantic and figural memory and verbal abstracting functions were included in a correlational analysis. No significant correlations between N1 amplitude or latency and neuropsychological tests were obtained. However, visual Nd amplitude correlated significantly with perceptual-motor tests and figural memory scores in the alcoholics. Significant correlations were found in alcoholics for visual P3 amplitude at PZ and delayed figural memory scores and two of the perceptual-motor tests. No significant correlations were obtained among the controls. These data indicate that significant relationships exist between some neuropsychological and ERP measures but that these relationships are restricted to measures of perceptual-motor functioning and to delayed figural memory.

Adult↗

PubMed1988

Human-milk intake measured by administration of deuterium oxide to the mother: a comparison with the test-weighing technique.

A comparison was made between the dose-to-the-mother deuterium-dilution method and the conventional test-weighing technique for determining human-milk intake in five exclusively breast-fed infants and in four breast-fed infants who received supplemental foods. After administration of 2H to the mothers human milk and infant urine were sampled over 14 d and analyzed for 2H:1H ratios by gas-isotope-ratio mass spectrometry. Infant total body water was determined by 18O dilution. The test-weighing procedure was conducted for 5 d consecutively. The intake of human milk (mean +/- SD) estimated by 2H dilution was 648 +/- 63 g/d and estimated by test-weighing was 636 +/- 84 g/d. The mean difference between the two methods was not significantly different from 0. The 2H-dilution and test-weighing techniques provide similar estimates of human-milk intake.

Adult↗

PubMed1988

Interpersonal problem solving in alcoholics.

In Study 1, a verbal role-playing test of interpersonal problem solving, the Adaptive Skills Battery (ASB), and selected "impersonal" neuropsychological problem-solving tests were given to male alcoholics (N = 73) in a VA alcohol treatment program and to male nonalcoholic controls (N = 36). Alcoholics' ASB competency scores under a "give your typical response" instructional set were significantly lower than controls' scores. In response to a "give the very best response" set, ASB scores for alcoholics and controls did not differ. Alcoholics' interpersonal problem-solving deficits appear to be due to their less effective execution of cognitive or problem-solving skills as opposed to their capacities for such skills. A lack of correlation between performance on impersonal neuropsychological tests and performance on the ASB suggests that these areas of functioning involve quite different psychological processes. In Study 2, alcoholics with alcoholic family members (family history positive) were found to have lower ASB scores than family history negative alcoholics. "Best" ASB responses were positively correlated with therapists' ratings of treatment behaviors and treatment outcome; "typical" ASB responses were not. Thus, alcoholics' therapeutic progress was predicted by their conceptualization of the "best" response in the interpersonal problem-solving situations and not by their typical pretreatment response.

Adult↗

PubMed1988

Carbon kinetics of milk formation in Holstein cows in late lactation.

Carbon transfer to milk in Holstein cows in late lactation was measured by introducing changes in the natural stable carbon isotope composition of the feed. Six Holstein cows in mid-lactation were placed on a diet naturally low in 13C (-25.0% vs Pee Dee belemnite [PDB] an international carbon isotope standard), based on alfalfa-barley, and six others were placed on a diet naturally enriched in 13C (-11.5% vs PDB), based on corn. After a 7-wk equilibration period on these diets, three cows were switched from alfalfa-barley to corn, and three were switched from corn to alfalfa-barley. The three other cows in each group served as controls. 13C/12C ratios were measured in daily morning milk samples during the week before and for 6 wk after the changes in diet. After the diets had been switched, milk isotope ratios rapidly approached the isotopic composition of the new diet, indicating rapid transfer of dietary carbon into milk. The data were consistent with a model whereby milk was synthesized from a single precursor pool that responded rapidly to dietary perturbation. The milk precursor pool had a half-life of approximately .9 d and had a mass of approximately 7 kg of carbon, which was renewed daily by the entry of 5 kg of digestible dietary carbon.

Animals↗

PubMed1987

Structural studies of apolipoprotein B: physical properties of the protein in guanidine hydrochloride.

Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (Mz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540,000 +/- 110,000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25 degrees C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640,000 and 250,000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.

Apolipoproteins B↗

PubMed1986

Self-association and phospholipid binding properties of iodinated apolipoprotein A-I.

Kinetic turnover studies of apolipoprotein metabolism often utilize radioiodinated tracers. These studies rely on the "tracer assumption" that the modified tracer is physiologically and metabolically identical with the native unmodified tracer. This paper addresses the validity of this assumption on the basis of the examination of the state of self-association and binding properties with egg yolk phosphatidylcholine small unilamellar vesicles of native and iodinated apolipoprotein A-I (apoA-I). Human apoA-I was iodinated to the extent of 1.0 and 3.7 mol of nonradioactive iodine/mol of protein. At concentrations from 0.013 to 0.8 mg/mL, iodinated apoA-I underwent concentration-dependent self-association similar to that of native apoA-I as evidenced by circular dichroism and gel filtration. At all concentrations, however, the iodinated preparations were more highly self-associated as judged by gel filtration in relation to the extent of iodination. Scatchard analysis of fluorometric titrations of apoA-I/vesicle interactions demonstrated that the binding capacity of vesicles for apoA-I increased and apoA-I binding affinity decreased upon iodination. In addition, the kinetics of apoA-I binding to vesicles was enhanced by iodination. The affinity, capacity, and kinetics of apoA-I binding were each altered 2-3-fold dependent on the extent of iodination. Since the dynamic interactions of apoA-I are perturbed by iodination, one may legitimately question whether the "tracer assumption" is valid for 125I-apoA-I under all experimental conditions.

Apolipoprotein A-I↗

PubMed1986

Immunologic comparison of the conformations of apolipoprotein B. Investigation of methodologies for the reconstitution of delipidated and denatured apolipoprotein B with nonionic surfactants.

Immunologic probes have been used to examine the conformation of apolipoprotein B (apo-B) as it exists within native low density lipoprotein (LDL) after lipid displacement with Triton X-100 and after denaturation with guanidine hydrochloride organic solvent delipidation and reconstitution with Triton X-100. Antigenic expression was assayed in two systems: by using either Triton X-100 or bovine serum albumin to maintain protein solubility. Apo-B delipidated by lipid displacement using Triton X-100 was virtually identical to LDL-apo-B in both systems, as assayed by polyclonal antisera prepared in rabbits against either antigen. Thus the native antigenic sites are preserved, although the displacement of the lipid core of LDL drastically alters the physical properties of the particle. Apo-B delipidated by solvent extraction in guanidine was reconstituted with Triton X-100 by several methods, and the products were examined immunologically. One method yielded a product that resembled apo-B as delipidated with Triton X-100, although full reconstitution could not be achieved. Nevertheless, Triton promoted refolding of apo-B to reform partial native structure as judged immunologically. By using both physical and immunologic methods for assessing structure, it is clearly evident that the perceptions of the conformational states of reconstituted apo-B can be very different, and multiple criteria need to be used to assess lipoprotein reconstitution.

Antigen-Antibody Complex↗

PubMed1986

Kinetic model of whole-body vanadium metabolism: studies in sheep.

A compartmental model for vanadium metabolism in sheep has been proposed. The model is consistent with data obtained from sheep fed a control diet (2.6 ppm vanadium) containing 0 or 200 ppm supplemental vanadium. Sheep were administered 48V dioxovanadium either orally or intravenously. Blood, feces, and urine radioactivity were monitored for 6 days postdosing. Several new insights regarding vanadium metabolism are suggested and tested against the data using the model. Some of these include significant absorption of 48V occurs from the upper gastrointestinal tract; an in vivo process is necessary in order for 48V dioxovanadium to be converted into a more biologically reactive species; at steady state the upper and lower gastrointestinal tracts contain at least 10- and 100-fold more mass of vanadium, respectively, than does blood. No statistically significant differences in transport rate constants were found between animals receiving 0 and 200 ppm supplemental dietary vanadium. The availability of a model will enable the refinement of future studies regarding vanadium metabolism in the ruminant.

Administration, Oral↗

PubMed1986

Vanadium metabolism in sheep. III. Influence of dietary vanadium on kinetics of 48V administered orally or intravenously and comparison of compartmental and graphical models.

Radiotracer techniques were used to investigate the influence of dietary stable V on the excretion, distribution and blood clearance kinetics of 48V in 14 rams averaging 58 kg body weight. Rams were fed a basal diet with added levels of 0, 50 or 200 mg/kg V as NH4 VO3 for 25 wk before either oral or iv administration of the isotope. A three-compartment model was determined by graphical logarithmic analysis of blood disappearance data from iv-dosed rams and compared with a simultaneous multicompartment model, which made it possible to ascribe physiological processes to the components of the graphical model. The principal route of excretion of 48V administered iv was via urine, whereas the isotope given orally was excreted almost entirely by way of feces, resulting in low tissue and urinary 48V levels. Increasing dietary V increased (P less than .05) the percentage of dose excreted in urine regardless of dosing route, but dietary V had no effect on 48V excreted in feces. Stable dietary V had no effect on blood clearance rates of orally or iv-dosed rams. Dietary V addition decreased 48V concentration in kidney (P less than .01), liver, spleen, testes and muscle (P less than .05) of iv-dosed rams, but had no effect in rams dosed orally. Kidney, bone, liver and spleen retained the highest levels of 48V activity 144 h after dosing. Dietary V appeared to have a minimal effect on V kinetics in rams.

Administration, Oral↗

PubMed1985

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay.

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.

Apolipoproteins B↗

PubMed1985

Immune complex hyperlipidemia induced by an apolipoprotein-reactive immunoglobulin A paraprotein from a patient with multiple myeloma. Characterization of this immunoglobulin.

An antibodylike paraprotein has been isolated from a patient with multiple myeloma and autoimmune hyperlipoproteinemia. The paraprotein bound to apolipoprotein B (apo B)-containing lipoproteins that formed macromolecular aggregates, and globules thought to be aggregated complexes of lipoproteins and reactive immunoglobulins were observed circulating within the retinal blood vessels of this patient. This binding specificity permitted purification of the paraprotein from both the agglutinated immune complexes and from the plasma. The protein is an IgA, kappa-immunoglobulin which exists primarily in a polymeric state. Capillary immunoprecipitation demonstrated reactivity with very low density lipoproteins (VLDL) and low density proteins (LDL), but not with high density lipoproteins (HDL). Delipidated apo B and apo E, but not apo A or apo C, formed precipitates with this immunoglobulin. In using a radioimmunoassay format, the affinity of the immunoglobulin was greatest for VLDL and decreases sequentially for intermediate density lipoproteins and LDL. No binding occurred with a dispersion of LDL lipids or with HDL. Deglycosylation did not change the binding to LDL. The apolipoproteins B and E bound with similar affinity, but no binding occurred with apo A-I or apo A-II. Weak binding appeared to occur with apo C. This paraprotein immunoprecipitated apo B-containing lipoproteins from all classes of vertebrates tested. Displacement of the lipids of LDL by Triton X-100 resulted in the formation of an apo B-Triton complex which, however, did not bind to the immunoglobulin; apparently the binding site on apo B was lost. Upon enzymatic digestion with the IgA-specific protease from Streptococcus sanguis the immunoglobulin was cleaved into Fc and Fab fragments, and the binding of LDL occurred only with the latter, consistent with the behavior of an immunoglobulin. The immunoreactivity of this paraprotein with apo B and apo E raises the interesting possibility that it may be binding to a site on these apolipoproteins which is reactive with the apo B, E receptor of the plasma membrane, a site which is conserved throughout the vertebrate phylum.

Aged↗

PubMed1984

A method for the measurement of the sedimentation coefficient and molecular weight of microgram quantities of proteins in 6 M guanidine hydrochloride.

A technique has been perfected for measuring the sedimentation coefficient of microgram quantities of a reduced protein in 6 M guanidine hydrochloride. The protein is sedimented through a gradient of 5-8 M guanidine-HCl in the presence of dithiothreitol in a SW 50.1 swinging-bucket rotor. Run conditions are calibrated by a simultaneous measurement using a single reference protein. Thus, the need for running a calibration curve involving several standard proteins simultaneously with a sample is eliminated. Because of the trace quantity of protein used, the technique yields an estimate of the sedimentation coefficient at zero concentration (s0) directly without extrapolation. Since s0 is a function of the molecular weight of a reduced protein in this solvent, the method also allows an estimate of the subunit molecular weight of the protein. The results of the application of the method to known proteins are reported.

Centrifugation, Density Gradient↗

PubMed1984

Structural studies on apolipoprotein B: controllable heterogeneity of the complex formed with the surfactant, Triton X-100.

Apolipoprotein B complexed with Triton X-100 (T-ApoB) has been isolated from human low density lipoprotein (LDL). Preparations are heterogeneous when analyzed by sedimentation velocity, with a major 12 S species and minor 17 S species present. The 12 S T-ApoB complex possesses a molecular weight of 880,000 containing 400,000 daltons of protein. Hydrodynamic measurements on this complex are consistent with a prolate ellipsoid model having an axial ratio of 13:1 and 0.22 g/g of bound water. Heterogeneity results from the irreversible aggregation of 12 S complexes into discrete 17 S and faster sedimenting components. A significant finding is that three determinants of this T-apoB heterogeneity could be elucidated and controlled. First, the initial state of aggregation is mainly influenced by the technique by which Triton and LDL are mixed. Second, once isolated, T-ApoB complexes slowly but spontaneously undergo further aggregation at 4 degrees C; the rate and extent of aggregation is enhanced remarkably with increasing temperature. Finally, reagents that unfold and expose protein structure (perchlorate, thiocyanate, and reducing reagents) lead to increased aggregation. The ability to control heterogeneity carries important implications for other studies concerning interactions of apoB with surfactants and lipids.

Apolipoproteins B↗

PubMed1983

A comparative study of enzymatic digestion profiles of apolipoprotein B from four human subjects.

A methodological approach for comparative structural study of apolipoprotein B has been developed. Low-density lipoproteins from four human subjects were digested in three separate enzyme systems, utilizing trypsin, chymotrypsin and Staphylococcus aureus protease V8, each in the presence of 1% sodium dodecyl sulfate. The peptides were separated by electrophoresis on polyacrylamide gels in SDS; the stained gels were scanned spectrophotometrically to produce characteristic profiles. Comparison of the profiles revealed good reproducibility and a high degree of similarity among the different subjects. Of the four subjects studied, one subject had one apparent difference in the tryptic digest profile and also in the S. aureus protease V8 digest profile. The structural significance of these variations can be evaluated only after a larger number of subjects, including those presented here, have been examined; this study is now in preparation.

Apolipoproteins↗