Reproducibility of glucose, fatty acid and VLDL kinetics and multi-organ insulin sensitivity in obese subjects with non-alcoholic fatty liver disease.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is associated with abnormalities in basal glucose and free fatty acid (FFA) metabolism, multi-organ insulin resistance and alterations in lipoprotein kinetics. These metabolic outcomes can be evaluated in vivo by using stable isotopically labeled tracer methods. An understanding of the reproducibility of these measures is necessary to ensure adequate statistical power in studies designed to evaluate metabolic function in subjects with NAFLD. METHODS: We determined the degree of intra-individual variability of skeletal muscle, adipose tissue, and hepatic insulin sensitivity and basal plasma glucose, FFA, and very-low-density lipoprotein triglyceride and apolipoprotein B-100 (apoB-100) kinetics in eight obese subjects with NAFLD (age: 44 ± 3 years; body mass index: 38.2 ± 1.7 kg m(-2); intrahepatic triglyceride content: 24.5 ± 3.9%), by using the hyperinsulinemic-euglycemic clamp technique and stable isotope-labeled tracer methods and mathematical modeling on two separate occasions ∼2 months apart. RESULTS: The intra-individual variability (coefficient of variation) ranged from 6% for basal glucose production to 21% for insulin-stimulated glucose disposal (percentage increase from basal). We estimated that a 25% difference in any outcome measure can be detected with a sample size of ≤ 8 subjects for paired studies and ≤ 15 subjects per group for unpaired studies, assuming an α value of 0.05 and a ß value of 0.20 (that is, 80% power). CONCLUSION: These results demonstrate that only a small number of subjects are needed to detect clinically relevant effects in insulin sensitivity and hepatic lipoprotein metabolism in obese subjects with NAFLD, and will be useful to determine appropriate sample size for future metabolic studies.

Decrease in hepatic very-low-density lipoprotein-triglyceride secretion after weight loss is inversely associated with changes in circulating leptin.

AIM: Although weight loss usually decreases very-low-density lipoprotein-triglyceride (VLDL-TG) secretion rate, the change in VLDL-TG kinetics is not directly related to the change in body weight. Circulating leptin also declines with weight loss and can affect hepatic lipid metabolism. The aim of this study was to determine whether circulating leptin is associated with weight loss-induced changes in VLDL-TG secretion. METHODS: Ten extremely obese subjects were studied. VLDL-TG secretion rate and the contribution of systemic (derived from lipolysis of subcutaneous adipose tissue TG) and non-systemic fatty acids (derived primarily from lipolysis of intrahepatic and intraperitoneal TG, and de novo lipogenesis) to VLDL-TG production were determined by using stable isotopically labelled tracer methods before and 1 year after gastric bypass surgery. RESULTS: Subjects lost 33 +/- 12% of body weight, and VLDL-TG secretion rate decreased by 46 +/- 23% (p = 0.001), primarily because of a decrease in the secretion of VLDL-TG from non-systemic fatty acids (p = 0.002). Changes in VLDL-TG secretion rates were not significantly related to reductions in body weight, body mass index, plasma palmitate flux, free fatty acid or insulin concentrations. The change in VLDL-TG secretion was inversely correlated with the change in plasma leptin concentration (r = -0.72, p = 0.013), because of a negative association between changes in leptin and VLDL-TG secretion from non-systemic fatty acids (r = -0.95, p < 0.001). CONCLUSIONS: Weight loss-induced changes in plasma leptin concentration are inversely associated with changes in VLDL-TG secretion rate. Additional studies are needed to determine whether the correlation between circulating leptin and VLDL-TG secretion represents a cause-and-effect relationship.

Impaired plasma nonesterified fatty acid tolerance is an early defect in the natural history of type 2 diabetes.

CONTEXT: Abnormal plasma nonesterified fatty acid (NEFA) metabolism may play a role in the development of type 2 diabetes. OBJECTIVES: Our objectives were to demonstrate whether there is a defect in insulin-mediated suppression of plasma NEFA appearance (RaNEFA) and oxidation (OxNEFA) during enhanced intravascular triacylglycerol lipolysis early in the natural history of type 2 diabetes, and if so, to determine whether other mechanisms than reduced insulin-mediated suppression of intracellular lipolysis are involved. DESIGN: These are cross-sectional studies. SETTING: The studies were performed at an academic clinical research center. PARTICIPANTS: Nine healthy subjects with both parents with type 2 diabetes (FH+) and nine healthy subjects with no first-degree relatives with type 2 diabetes (FH-) with similar anthropometric features were included in the studies. INTERVENTIONS: Pancreatic clamps and iv infusion of stable isotopic tracers ([1,1,2,3,3-(2)H5]-glycerol and [U-(13)C]-palmitate or [1,2-(13)C]-acetate) were performed while intravascular triacylglycerol lipolysis was simultaneously clamped by iv infusion of heparin plus Intralipid at low (fasting) and high insulin levels. Oral nicotinic acid (NA) was used to inhibit intracellular lipolysis. MAIN OUTCOME MEASURES: RaNEFA and OxNEFA were determined. RESULTS: During heparin plus Intralipid infusion at high plasma insulin levels, and despite similar intravascular lipolytic rates, FH+ had higher RaNEFA and OxNEFA than FH- (RaNEFA: 17.4+/-6.3 vs. 9.2+/-4.2; OxNEFA: 4.5+/-1.8 vs. 2.3+/-1.5 micromol/kg lean body mass/min), independent of NA intake, gender, age, and body composition. In the presence of NA, insulin-mediated suppression of RaNEFA was still observed in FH-, but not in FH+. CONCLUSIONS: Increased RaNEFA and OxNEFA during intravascular lipolysis at high insulin levels occur early in the natural history of type 2 diabetes.

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.

Bovine apolipoproteins C. I. Isolation and spectroscopic investigations of the phospholipid binding properties.

Seven low-molecular weight proteins of the C class of apolipoproteins have been isolated from bovine serum high density lipoprotein. Amino acid analysis has shown five of these to be equivalent to the apolipoproteins previously described (Lim, C.T. and Scanu, A.M. (1976) Artery 2, 483-496). A spectroscopic examination of these proteins in the presence of increasing amounts of L-alpha-dimyristoyl phosphatidylcholine single-bilayer vesicles indicates that all bovine apolipoproteins C exhibit changes in secondary and tertiary structure as shown by intrinsic fluorescence intensity, wavelength, and polarization changes, and increases in alpha-helical content as seen by circular dichroism. Evidence is presented to show that bovine apolipoproteins C, like all human apolipoproteins of the A and C classes, can cause phospholipid multilamellar liposomes to disrupt and/or rearrange into a smaller complex which scatters less light. This paper details the screening of the bovine apolipoproteins for their phospholipid binding properties, whereas the following paper will examine the nature of their complexes with phospholipid in more detail. Together, these papers represent the first investigation of protein-lipid interactions involving any nonhuman apolipoproteins C.

Bovine apolipoproteins C. II. Isolation and partial physicochemical characterization of complexes with L-alpha-dimyristoyl phosphatidylcholine.

Complexes were made between L-alpha-dimyristoyl phosphatidylcholine (DMPC) and four of the seven bovine apolipoproteins C described in the preceding paper (Patterson, B.W. and Jonas, A. (1980) Biochim. Biophys, Acta 619, 572-586). Reaction mixtures were fractionated by gel filtration chromatography and isopycnic density gradient ultracentrifugation. Selected complexes were further analyzed by sedimentation equilibrium ultracentrifugation and examined for phospholipid bilayer phase transition properties as reported by the fluorescence polarization of a lipophilic probe. Two bovine apolipoproteins C (D2, D3) were able to form complexes with DMPC of virtually the same size, stoichiometry, and density over a wide range of initial lipid/protein molar ratios (200 : 1 to 10 : 1). At very high initial molar ratios (200 : 1 and 100: 1), an additional lipid-enriched complex was formed with these apolipoproteins. Complexes made with D4 were less discrete, having a size, stoichiometry, and density dependent on the initial lipid : protein ratios used. Isolated complexes were smaller than intact DMPC vesicles, representing a break-down product of vesicular structure. The sizes (molecular weights around 2-3 x 10(5)), hydrated densities (1.06-1.12 g/ml), and weight percentage protein compositions (30-40%) of bovine C apolipoprotein-DMPC complexes are comparable to the corresponding parameters for intact bovine HDL. Isolated bovine C apolipoprotein-DMPC complexes retain some phospholipid bilayer structure as indicated by their phase transition behavior. However, the phase transition is considerably broadened and shifted to a higher temperature in the complexes compared to pure lipid. The results obtained are comparable to the phospholipid binding properties of human C apolipoproteins and are consistent with various oblate ellipsoidal models suggested for apolipoprotein-DMPC complexes.

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.

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.

The effect of systemic versus portal insulin delivery in pancreas transplantation on insulin action and VLDL metabolism.

Combined kidney-pancreas transplantation (KPT) with anastomosis of the pancreatic vein to the systemic circulation (KPT-S) or to the portal circulation (KPT-P) provides a human model in which the chronic effects of portal versus systemic insulin delivery on glucose and VLDL metabolism can be examined. Despite similar plasma glucose and C-peptide levels, KPT-S (n = 9) had an approximate twofold elevation of fasting and intravenous glucose-stimulated plasma insulin levels compared with both KPT-P (n = 7) and healthy control subjects (n = 15). The plasma free fatty acid (FFA) levels were elevated in both transplant groups versus control subjects, but the plasma insulin elevation necessary to lower plasma FFA by 50% was approximately two times higher in KPT-S versus KPT-P and control subjects. Endogenous glucose production was similar in KPT-S and KPT-P, despite approximately 35% higher hepatic insulin levels in the latter, and was suppressed to a greater extent during a euglycemic-hyperinsulinemic clamp in KPT-S versus KPT-P. Total-body glucose utilization during the euglycemic-hyperinsulinemic clamp was approximately 40% lower in KPT-S versus KPT-P, indicating peripheral tissue but not hepatic insulin resistance in KPT-S versus KPT-P. Both transplant groups had an approximate twofold elevation of triglyceride (TG)-rich lipoprotein apolipoprotein B (apoB) and lipids versus control subjects. Elevation of VLDL-apoB and VLDL-TG in both transplant groups was entirely explained by an approximately 50% reduction in clearance of VLDL compared with healthy control subjects. In the presence of increased FFA load but in the absence of hepatic overinsulinization and marked hepatic insulin resistance, there was no elevation of VLDL secretion in KPT-S versus KPT-P and control subjects. These findings suggest that chronic systemic hyperinsulinemia and peripheral tissue insulin resistance with the consequent elevation of plasma FFA flux are insufficient per se to cause VLDL overproduction and that additional factors, such as hepatic hyperinsulinemia and/or gross insulin resistance, may be an essential prerequisite in the pathogenesis of VLDL overproduction in the common form of the insulin resistance syndrome.

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.

Use of stable isotopically labeled tracers for studies of metabolic kinetics: an overview.

Stable isotopically labeled tracers offer a reliable and safe alternative to the use of radioactive tracers for studies of metabolic kinetics. This overview examines some of the principles and technical issues regarding mass spectrometry instrumentation, and reviews some of the approaches used in the application of stable isotopically labeled tracers to studies of protein, lipid, and carbohydrate metabolic kinetics.

Improved accuracy and precision of gas chromatography/mass spectrometry measurements for metabolic tracers.

The use of stable-isotope tracer methodology to study substrate metabolic kinetics requires accurate measurement of the tracer to tracee ratio (TTR), often by gas chromatography/mass spectrometry (GC/MS). Many approaches for measurement of the TTR by GC/MS do not use standards of known isotopic enrichment to control for variability in instrument response. In addition, most GC/MS applications exhibit some degree of concentration dependency whereby the measured ion abundance ratio varies with the quantity of sample analyzed, thereby placing a limitation on the accuracy of isotopic enrichment standard curves unless the quantities of standards and samples analyzed are closely matched. We document the degree to which day-to-day variability can affect the instrument response for several GC/MS analyses of metabolic tracers when isotopic enrichment standards are not used to control for variable instrument response. Furthermore, we report a new approach that incorporates concentration dependencies within a standard curve to improve the accuracy and precision of TTR measurements over a range of sample quantities analyzed. The new approach was applied to plasma samples obtained from experimental protocols performed in human subjects with three commonly used tracers: 2H2-palmitate, 15N2-urea, and 13C-leucine. Variability in the day-to-day instrument response was 84% and 26% for 2H2-palmitate and 15N2-urea, respectively; in addition, up to 10% variability due to concentration dependency was noted for these applications. The new approach virtually eliminated these sources of variability. After controlling for concentration dependency, a threefold reduction in the standard error was noted when the enrichment of 13C-leucine measured by electron-impact (EI) ionization GC/MS was correlated against negative chemical ionization (NCI) GC/MS. These data demonstrate that our new approach decreases the errors in TTR determination caused by variations in instrument response and concentration dependency. This approach is generically applicable, and can improve the accuracy and precision of TTR determinations for most GC/MS analyses.

Urea and protein metabolism in burned children: effect of dietary protein intake.

The response of urea metabolic kinetics, the rate of whole-body protein breakdown, and muscle and skin protein synthesis rates to dietary protein intake (1.15 to 2.92 g/kg/d) was assessed in children with 20% to 40% total body surface area burn injury using a primed continuous infusion of 15N2-urea and L-13C6-phenylalanine. Plasma urea concentration, production, and excretion rates increased with dietary protein intake without evidence of approaching maximum plateau values. There was no consistent evidence of urea recycling in these subjects (urea production = excretion) at any level of protein intake. The rate of appearance (Ra) of phenylalanine (an index of whole-body protein breakdown) and rate of muscle protein synthesis were independent of dietary protein, whereas there was a significant increase in skin protein synthesis with higher protein intake. We conclude that there seems to be little benefit of high protein intake on whole-body protein breakdown and muscle protein synthesis rates in these burn patients, although high-protein diets may enhance wound healing.

Measurement of very low stable isotope enrichments by gas chromatography/mass spectrometry: application to measurement of muscle protein synthesis.

Measurement of muscle protein synthesis using stable isotopically labeled tracers usually requires isotope ratio mass spectrometry (IRMS) because of the need to measure very low enrichments of stable isotopically labeled tracers (tracer to tracee ratio [TTR], 0.005% to 0.10%). This approach is laborious, requiring purification of the metabolite of interest and combustion to a gas for IRMS analysis, and is best suited for use with 13C tracers. We have developed an approach whereby low enrichments can be conveniently measured by a conventional gas chromatography/mass spectrometry (GC/MS) instrument. The approach includes three critical elements: (1) use of a highly substituted tracer containing three or more labeled atoms, to measure enrichment above a very low natural abundance of highly substituted isotopomers; (2) use of a highly substituted natural abundance isotopomer as a base ion for comparison rather than the most abundant m + 0 isotopomer, to reduce the dynamic range of the isotopomer ratio measurement; and (3) a sensitive mass spectrometric analysis that measures the natural abundance of the isotopomer used as a tracer with a high signal to noise ratio (> 100:1). This approach was used to measure the rate of synthesis of muscle protein following a primed continuous infusion of L-[13C6]-phenylalanine (PHE) in eight fasted dogs and L-[2H3]-leucine in five fasted human subjects. Values for [13C6]-PHE enrichment by GC/MS rates were virtually identical to those obtained by a conventional approach using high-performance liquid chromatography (HPLC) to isolate PHE, combustion to CO2, and measurement of 13CO2 enrichment by IRMS (IRMS enrichment = 0.9988 x GC/MS enrichment, R2 = .891), resulting in identical values for muscle fractional synthesis rates ([FSRs] mean +/- SEM: 2.7 +/- 0.2 and 2.5 +/- 0.2%/d for GC/MS and IRMS, respectively). Human muscle synthesis rates measured by GC/MS analysis of [2H3]-leucine enrichment (1.90 +/- 0.17%/d) were similar to published values based on IRMS analysis using a 1- 13C-leucine tracer. We conclude that compared with the IRMS approach, the GC/MS approach offers faster throughput, has a lower sample requirement, and is suitable for a wider variety of tracers such as 2H. The principles outlined here should be applicable to the measurement of low enrichments by GC/MS in a wide variety of stable isotope tracer applications.

A new apolipoprotein B truncation (apo B-43.7) in familial hypobetalipoproteinemia: genetic and metabolic studies.

We describe a new truncation of apolipoprotein (apo) B in a white kindred with familial hypobetalipoproteinemia (FHBL). Apo B-43.7, found in a daughter and her father, was due to a C --> T change in base position 6162 of the apo B gene converting the arginine (residue 1986) codon CGA to a stop codon TGA. Both subjects were heterozygotes, and both apo B-43.7- and apo B-100-containing particles were present in plasma. On density gradient ultracentrifugation (DGUC), approximately 30% to 40% of apo B-43.7 floated with very-low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)-density particles and 60% to 70% floated with high-density lipoprotein (HDL)-density particles. To assess the metabolism of apo B, 13C-leucine was infused and its rates of appearance in and disappearance from apo B-43.7- and apo B-100-containing particles were quantified by multicompartmental kinetic analysis. Apo B-100 entered plasma via VLDL with a production rate of 30 mg x kg-1 x d-1. Fractional catabolic rates (FCRs) for apo B-100 VLDL, IDL, and low-density lipoprotein (LDL) were 20.0, 16.0, and 0.46 pools x d-1, respectively. The production rate of apo B-43.7 was 9.6 mg x kg-1 x d-1, and FCRs for apo B-43.7 VLDL- and HDL-like particles were 12.0 and 1.8 pools x d-1, respectively. Approximately 30% of apo B-43.7 in HDL-density particles was derived from VLDL apo B-43.7, and about 70% appeared to enter the plasma as HDLs. The relatively low production rate of apo B-43.7 is compatible with previous reports that apo B truncations are produced at lower rates than their apo B-100 counterparts.

Use of stable isotope labeling technique and mass isotopomer distribution analysis of [(13)C]palmitate isolated from surfactant disaturated phospholipids to study surfactant in vivo kinetics in a premature infant.

Pulmonary surfactant is a complex mixture of phospholipids and proteins which lowers surface tension and maintains alveolar expansion at end expiration. Developmental and genetic disruption of pulmonary surfactant metabolism leads to respiratory distress in newborns. Stable isotope labeling of metabolic precursors of disaturated phospholipids, the most abundant and specific component of pulmonary surfactant, permits the measurement of the kinetics of surfactant metabolism in vivo. We measured [U-(13)C(6)]glucose incorporation into palmitic acid derived from disaturated surfactant phospholipids. A 24 h infusion of [U-(13)C(6)]glucose (140 mg kg(-1)) was administered to a premature infant who required mechanical ventilation for respiratory distress syndrome; tracheal aspirate samples were obtained at the start of the infusion and at regular intervals for the next 70 h. Each tracheal aspirate sample was incubated with osmium tetroxide to isolate disaturated surfactant phospholipids. Methyl esters of the fatty acids in the disaturated phospholipids were prepared and the enrichment of [(13)C]methyl palmitate was measured by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combination/isotope ratio mass spectrometry (GC/C/IRMS). Mass isotopomer distribution analysis (MIDA) was used to calculate the fractional synthetic rate (FSR) of palmitate synthesized from acetate. With both GC/MS and GC/C/IRMS, palmitate (13)C enrichment was first detected 12.3 h after the start of the tracer infusion. The enrichment increased in a linear fashion, reached a peak at 47 h and remained constant in the remainder of the samples. The FSR of palmitate from acetate was 5.2% per day. Stable isotope techniques and MIDA will provide insights into the kinetics of surfactant metabolism in newborns with respiratory dysfunction.

Alcoholism and family history of alcoholism: effects on visual and auditory event-related potentials.

In a dual-modality paradigm, visual and auditory event-related potentials were elicited in 40 alcoholic men and 30 controls, equated with the alcoholics on age and education. Half of each group had first-degree relatives who were alcoholic (family history positive). The amplitude of the visual N1 component was reduced among the alcoholics, but their auditory N1 amplitudes were normal. Average N1 amplitudes were also smaller in the family history positive subjects but this effect was significant only for auditory stimuli. Alcoholics showed reduced average P3 amplitudes to both visual and auditory signals, particularly in the family history positive group. Clearly, stratification by family history is useful for ascertainment of ERP variation among alcoholics. There were no effects on P3 latency. Among several possible explanations of P3 deficits in alcoholics, two are particularly interesting: (1) alcoholics cannot mobilize sufficient processing resources in the service of effortful cognitive functions; (2) alcoholics, being poorly motivated, apply insufficient effort to cognitive tasks. An experiment designed to test these hypotheses is described.

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.

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.