Differences in Gut Metabolites and Microbial Composition and Functions between Egyptian and U.S. Children Are Consistent with Their Diets.

Previous studies indicated that populations consuming a Mediterranean diet rich in fiber, vegetables, and fruits have a significantly lower risk of cardiovascular and metabolic diseases than populations of industrialized societies consuming diets enriched in processed carbohydrates, animal proteins, and fats. To explore the potential contributions of gut microbiota to the observed diet-related metabolic effects, we conducted an integrative analysis of distal gut microbiota composition and functions and intestinal metabolites in Egyptian and U.S. teenagers. All Egyptian gut microbial communities belonged to the Prevotella enterotype, whereas all but one of the U.S. samples were of the Bacteroides enterotype. The intestinal environment of Egyptians was characterized by higher levels of short-chain fatty acids, a higher prevalence of microbial polysaccharide degradation-encoding genes, and a higher proportion of several polysaccharide-degrading genera. Egyptian gut microbiota also appeared to be under heavier bacteriophage pressure. In contrast, the gut environment of U.S. children was rich in amino acids and lipid metabolism-associated compounds; contained more microbial genes encoding protein degradation, vitamin biosynthesis, and iron acquisition pathways; and was enriched in several protein- and starch-degrading genera. Levels of 1-methylhistamine, a biomarker of allergic response, were elevated in U.S. guts, as were the abundances of members of Faecalibacterium and Akkermansia, two genera with recognized anti-inflammatory effects. The revealed corroborating differences in fecal microbiota structure and functions and metabolite profiles between Egyptian and U.S. teenagers are consistent with the nutrient variation between Mediterranean and Western diets. IMPORTANCE The human gastrointestinal microbiota functions as an important mediator of diet for host metabolism. To evaluate how consumed diets influence the gut environment, we carried out simultaneous interrogations of distal gut microbiota and metabolites in samples from healthy children in Egypt and the United States. While Egyptian children consumed a Mediterranean diet rich in plant foods, U.S. children consumed a Western diet high in animal protein, fats, and highly processed carbohydrates. Consistent with the consumed diets, Egyptian gut samples were enriched in polysaccharide-degrading microbes and end products of polysaccharide fermentation and U.S. gut samples were enriched in proteolytic microbes and end products of protein and fat metabolism. Thus, the intestinal microbiota might be selected on the basis of the diets that we consume, which can open opportunities to affect gut health through modulation of gut microbiota with dietary supplementations.

Total body water reference values and prediction equations for adults.

BACKGROUND: The clinical interpretation of total body water (TBW) necessitates the availability of timely comparative reference data. The prediction of TBW volume in renal disease is critical in order to prescribe and monitor the dose of dialysis in the determination of Kt/V. In clinical practice, urea distribution (V) is commonly predicted from anthropometric equations that are several decades old and for white patients only. This article presents new reference values and prediction equations for TBW from anthropometry for white and black adults. METHODS: The study sample included four data sets, two from Ohio and one each from New Mexico and New York, for a total of 604 white men, 128 black men, 772 white women, and 191 black women who were 18 to 90 years of age. The TBW concentration was measured by the deuterium or tritium oxide dilution method, and body composition was measured with a Lunar DXA machine. An all-possible-subsets of regression was used to predict TBW. The accuracy of the selected equations was confirmed by cross-validation. RESULTS: Blacks had larger TBW means than whites at all age groups. The 75th TBW percentile for whites approximated the TBW median for blacks at most ages. The white men and black men and women had the largest TBW means ever reported for healthy individuals. The race- and sex-specific TBW prediction equations included age, weight, and stature, with body mass index (BMI) substituted for weight in the white men. The root mean square errors (RMSEs) and standard errors for the individual (SEIs) ranged from approximately 3.8 to 5.0 L for the men and from 3.3 to 3.6 L for the women. In both men and women, high values of TBW were associated with high levels of total body fat (TBF) and fat-free mass (FFM). CONCLUSION: : TBW in these healthy adults is relatively stable through a large portion of adulthood. There are significant race and sex differences in TBW. These accurate and precise equations for TBW provide a useful tool for the clinical prediction of TBW in renal disease for white and black adults. These are the first TBW prediction equations that are specific for blacks.

NMR spectroscopic analyses of liver phosphatidylcholine and phosphatidylethanolamine biosynthesis in rats exposed to peroxisome proliferators-A class of nongenotoxic hepatocarcinogens.

Peroxisome proliferators (PPs) are commercial/industrial chemicals that display tumor promoter activity in rodents. The mechanism is not completely understood, and our ability to predict tumorigenicity a priori is even less developed. Wy-14,643, perfluorooctanoic acid (PFOA), and di(2-ethylhexyl)phthalate (DEHP) are strong, moderate, and weak tumor promoters, respectively, while perfluorodecanoic acid (PFDA) lacks promoter activity. This investigation examined the effects of these PPs on the biosyntheses of phosphatidylcholine (PtdC) and phosphatidylethanolamine (PtdE) in rat liver. After exposure to PPs, rats were administered [1-(13)C]choline + [2-(13)C]ethanolamine and liver extracts were analyzed by (31)P and (13)C NMR. The ratio of choline-derived to ethanolamine-derived phospholipids, R(c/e), was significantly affected by all PPs (p < 0. 05). R(c/e) values were in the order Wy-14,643 > PFOA > DEHP > control > PFDA. The amounts of PtdC derived via the CDP-choline pathway versus PtdE-N-methyltransferase (PEMT) activity was 71 vs 29% in controls. This distribution was significantly affected by treatments with Wy-14,643 (95 vs 5%), DEHP (87 vs 13%), and PFDA (39 vs 61%) (p < 0.02). Data suggest that Wy-14,643, PFOA, and DEHP cause a preference for choline and the CDP-choline pathway for biosynthesis of PtdC. Additionally, Wy-14,643 and DEHP inhibited the PEMT pathway. In contrast, PFDA-treated rats showed a preference for ethanolamine, and PtdC was predominately synthesized through the PEMT pathway. These data corroborate studies by Vance and co-workers which suggest that the pathways for PtdC biosynthesis are important for hepatocarcinogenesis. Further studies to evaluate the potential of these measurements as a biomarker for PP-associated tumorigenesis is warranted.

Dose-response hapatotoxicity of the peroxisome proliferator, perfluorodecanoic acid and the relationship to phospholipid metabolism in rats.

Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator that causes hepatotoxicity but lacks tumor-promoting activity in rats. We previously showed that a single dose of PFDA at 50 mg/kg (approximately LD50) causes an elevation in liver phosphocholine (PCho) and other effects related to phospholipid metabolism. In this study, we examined metabolic effects in the dose range 2-50 mg/kg in rats. At doses < or =20 mg/kg, PFDA is significantly less hepatotoxic than the LD50 as manifested by electron microscopy and measurements of daily food consumption and body weight. At 50 mg/kg rat serum tumor necrosis factor (TNF)-alpha concentration was increased 8-fold, while at 15 mg/kg there was no apparent increase in this cytokine. This lower dose, however, induces metabolic effects similar to those seen at the LD50. Liver fatty acyl-CoA oxidase activity showed a dose-dependent increase from 5-25 mg/kg PFDA. Treatments at 15 and 50 mg/kg caused a significant increase in liver phosphatidylcholine (28 and 66%) and phosphatidylethanolamine (31 and 74%). Both doses caused a significant increase in liver PCho but did not affect liver ATP levels, as manifested in 31P nuclear magnetic resonance (NMR) spectra from rat livers in vivo. These data suggest that the increase in liver [PCho] observed following PFDA exposure in rats represents a specific metabolic response, rather than a broad-range hepatotoxic effect.

Total body water data for white adults 18 to 64 years of age: the Fels Longitudinal Study.

BACKGROUND: Total body water (TBW) volume is reported to decrease with age, but much of the published data are 20 to almost 50 years old and are cross-sectional. Proper interpretation of clinical levels of TBW and trends with age necessitates the availability of current longitudinal data from healthy individuals. METHODS: Mixed longitudinal data for TBW of 274 white men and 292 white women (18 to 64 years of age) in the Fels Longitudinal Study were collected on a regular schedule over a recent eight-year period. The concentration of deuterium was measured by deuterium nuclear magnetic resonance spectroscopy. Body composition estimates were made with dual-energy x-ray absorptiometry, and random effect models were used to determine the patterns of change over time with and without covariates. RESULTS: The mean TBW data for the Fels men are either similar to or approximately 2 to as much as 6 liters greater than that reported by most other investigators 20 to 50 years ago. For Fels women, the mean TBW ranges from approximately 2 to as much as 5 liters less than that reported previously. These comparisons with much earlier studies reflect cohort effects and the secular changes in overall body size that have occurred during the past 60 to 70 years. These findings are reinforced by the fact that some early data sets included individuals born almost 140 years ago. After adjusting for the covariate effects of total body fat (TBF) and fat-free mass (FFM) with age, there were no significant age or age-squared effects on TBW in the men. In the women, after adjusting for the covariate associations of TBF and FFM with age, there was a small, but significant, negative linear association of TBW with age. In the men and women, the mean ratio of TBW to weight declined with age as a function of an increase in body fatness and more so for the men than the women. CONCLUSION: The findings from these mixed longitudinal data indicate that TBW volume, on average, maintains a reasonable degree of stability in men and women through a large portion of adulthood. These TBW data are recommended as current reference data for healthy adults.

Effects of peroxisome proliferators on rat liver phospholipids: sphingomyelin degradation may be involved in hepatotoxic mechanism of perfluorodecanoic acid.

Perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), clofibrate, di(2-ethylhexyl)phthalate (DEHP), and Wy-14,643 represent a class of compounds known as peroxisome proliferators (PPs). Such compounds induce biogenesis of liver peroxisomes and cause a varying degree of hepatotoxicity and carcinogenesis in rodents. We examined the effects of these PPs on rat hepatic lipids and phospholipid profiles using phosphorus-31 NMR spectroscopy. All PPs caused a 25-57% increase in hepatic phospholipid content, while all but clofibrate increased the total lipid content by 26-156%. Treatments also influenced the composition of liver phospholipids. Phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) contents were significantly increased in all treatment groups. Most notably, PFDA caused the largest increase in PtdCho and PtdEth content (ca. 70%), while PFOA and Wy-14,643 were the only test compounds that influenced the PtdCho:PtdEth ratio. PFDA also caused an ca. 30% decrease in sphingomyelin (SphM) from 24 to 120 h postdose. SphM is a key lipid in signal transduction processes involved in apoptosis. Hydrolysis of SphM can be mediated through the action of tumor necrosis factor (TNF-alpha). We measured the TNF-alpha concentrations in rat sera at 24 h post-PFDA-exposure and found an 8-fold increase relative to vehicle-treated controls. These data demonstrate that an increase in the serum TNF-alpha level correlates with the time frame for the observed reduction in hepatic SphM. PFOA, a structurally similar compound, had no effect on hepatic SphM content, nor did it affect the serum TNF-alpha concentration. These effects may be related to differences in the tumorigenicity associated with these compounds. We postulate that PFDA activates the SphM signal transduction pathway via the release of TNF-alpha. This then stimulates cytotoxic responses and processes of apoptosis and may suppress cell proliferative and mitogenic responses.

Perfluorodecanoic acid, a peroxisome proliferator, activates phospholipase C, inhibits CTP:phosphocholine cytidylyltransferase, and elevates diacylglycerol in rat liver.

Perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) are peroxisome proliferators that cause hepatotoxicity in rodents. This study shows that PFDA activates liver phospholipase C (PLC) and inhibits CTP:phosphocholine cytidylyltransferase (CT). PLC cytosolic and microsomal activities were increased 1.4- and 1.7-fold, respectively. CT activates were decreased to 58% (cytosol) and 36% (microsome) of control values. PFDA also caused a threefold increase in liver diacylglycerol (DAG) concentration. PFOA had no effect on the enzyme activities or DAG concentration. Together with previous results, these data suggest that PFDA activates a phosphatidylcholine-specific PLC causing an increase in liver phosphocholine and DAG. These effects are discussed in relation to cellular signalling processes that may provide a mechanism for PFDA-induced hepatotoxicity.

Influence of carbon chain length on the hepatic effects of perfluorinated fatty acids. A 19F- and 31P-NMR investigation.

Using nuclear magnetic resonance (NMR) spectroscopy, we investigated the importance of carbon chain length with regard to the hepatic effects associated with perfluoro-n-carboxylic acids. Male F-344 rats were administered a single intraperitoneal dose of either perfluoro-n-heptanoic acid (C7-PFA), perfluoro-n-nonanoic acid (C9-PFA), or perfluoro-n-undecanoic acid (C11-PFA). Data from previous studies involving perfluoro-n-octanoic acid (C8-PFA) and perfluoro-n-decanoic acid (C10-PFA) are included for comparison. Food consumption/body weight was monitored daily for all groups. C9- and C11-PFA treatment yields a prolonged hypophagic response while C7-PFA shows a more acute response. Fluorine-19 NMR spectra of urine and bile samples show no evidence of fluorometabolites and suggest that the distribution of perfluorocarbons into urine or bile is dependent upon carbon chain length. The aqueous solubility of C7-PFA appears to facilitate rapid urinary excretion, similar to that observed for C8-PFA. The relative hydrophobicity of C9- and C11-PFA appears to favor biliary enterohepatic recirculation, yielding a more protracted toxicity, similar to C10-PFA. Phosphorus-31 NMR studies of liver in vivo and liver extracts show that perfluorocarbons of > or = C9 carbons produce a significant increase in liver phosphocholine concentration. These data are discussed with regard to the impact of these chemicals on hepatic phospholipid metabolism. Hepatic peroxisomal fatty acyl CoA-oxidase activity (FAO) was measured to determine if C7-, C9-, and C11-PFA are peroxisome proliferators. Data indicate that the induction of peroxisomal enzyme activity by perfluorocarbons requires a chain length greater than seven carbons. In general, these results demonstrate the significance of carbon chain length in the hepatotoxic response and provide clues toward understanding the processes involved in the biological activities associated with exposure to these compounds.

Effect of the peroxisome proliferator perfluoro-n-decanoic acid on glucose transport in the isolated perfused rat liver.

The perfluorinated carboxylic acid, perfluoro-n-decanoic acid (PFDA), is a known peroxisome proliferator which displays toxicity in rodents. Using a paired-tracer first-pass extraction technique, the effect of PFDA on hepatic glucose transport was determined in the isolated perfused rat liver. In brief, livers isolated from PFDA-treated and control rats on day 5 posttreatment were administered the radiolabeled glucose analog, 3-O-[14C]methyl-D-glucose ([14C]3-O-MG) in addition to [fructose-1-3H(N)]sucrose ([3H]sucrose), which served as a measure of extracellular volume. Hepatic glucose transport was calculated from the change in the ratio [14C]3-O-MG/[3H]sucrose during passage through the liver. Data from this study indicate that PFDA inhibits hepatic glucose transport. Percent hepatic glucose extraction is 1.8-fold greater in controls than in PFDA-treated rats. No significant difference in lactate dehydrogenase levels was observed in the liver perfusate from PFDA-treated and control rats. This suggests that the difference in percent glucose extraction between PFDA-treated and control groups is specifically due to the PFDA treatment and is not attributed to differences in liver viability between groups. Although the exact mechanism for this inhibition in hepatic glucose transport is not known, it is hypothesized that PFDA may have a major impact on membrane structure/function which, in turn, may alter glucose transport.

Body composition in white adults by dual-energy x-ray absorptiometry, densitometry, and total body water.

Percent body fat (%BF) and fat-free mass (FFM) were determined in 151 adults from the Fels Longitudinal Study by total body scans with a Lunar DPX (DXA), by underwater weighing and residual lung volume with a two-compartment model using body density (Dens), and total body water (TBW) using deuterium dilution. For the DPX, the medium scan mode ensures precision up to 100 kg body wt and for a ratio of weight to stature < or = 0.7214. Because of these specifications, 15% (n = 23) of the original sample of 151 were excluded. Results for 78 women and 50 men are presented. %BFTBW was significantly (P < 0.05) less than %BFDens in women (mean difference 2.7 +/- 4.2%) and %BFDXA in men (mean difference 2.2 +/- 4.3%). No other significant intermethod differences were observed for %BF and FFM estimates. Pairwise regressions showed the lowest SEEs for %BFDens regressed vs %BFDXA (2.3% for men, 3.2% for women) and for FFMDens vs FFMDXA (2.2 kg for men, 1.9 kg for women). For each sex, reliability analyses and limits of agreement for body composition estimates showed less agreement between TBW and either Dens and DXA than between Dens and DXA.

Effects of perfluoro-n-octanoic acid, perfluoro-n-decanoic acid, and clofibrate on hepatic phosphorus metabolism in rats and guinea pigs in vivo.

Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy was used to study the effects of perfluoro-n-octanoic acid (PFOA), perfluoro-n-decanoic acid (PFDA), and clofibrate (CLOF) on liver phosphorus metabolism in rats and guinea pigs in vivo. All three compounds are known to cause peroxisome proliferation in rats but not in guinea pigs. The data indicate that indices related to overall tissue viability (i.e., adenosine triphosphate levels) remain unaffected at the doses and experimental times investigated for all treatments and both species. PFDA-treated rats revealed a marked increase in a liver phosphomonoester resonance compared with corresponding controls (p < or = 0.01); no such effect was observed in guinea pigs. This particular 31P NMR signal was identified as phosphocholine (PCho) and was found to steadily increase in concentration at consecutive days post-PFDA treatment, reaching 6.26 +/- 0.29 mumol/g liver at 5 days. This is fourfold greater than the PCho levels determined in livers from corresponding pair-fed control rats. The elevation in liver PCho is a specific response of PFDA treatment in rats and is not simply related to peroxisome proliferation in general, since neither PFOA nor CLOF produce such an effect. The data suggest a unique effect of PFDA on liver phospholipid metabolism, specifically phosphatidylcholine, which may involve enhanced phospholipid turnover via phosphatidylcholine-specific phospholipase C activity.

Effects of the peroxisome proliferator perfluoro-n-decanoic acid on hepatic gluconeogenesis and glycogenesis: a 13C NMR investigation.

Carbon-13 NMR spectroscopy was used to study the effects of the peroxisome proliferator perfluoro-n-decanoic acid (PFDA) on hepatic carbohydrate metabolism in male Fischer-344 rats. The data indicate that PFDA-treated rats display an inhibition in hepatic [1-13C]glucose and [3-13C]alanine utilization on day 5 posttreatment. PFDA rats show hepatic mean glucose and alanine intensities which are significantly greater (ca. 10-100%) than controls. With [1-13C]-glucose as substrate, PFDA rats show severe to complete inhibition in glycogenesis on days 3 and 5 posttreatment. With [3-13C]alanine as substrate, both groups show functional gluconeogenesis and glycogenesis; however, treated rats show a more transient and less intense C1-glycogen resonance relative to control. These data suggest that PFDA inhibits either the hepatocellular transport of glucose and/or its phosphorylation by glucokinase. The effect of PFDA on TCA cycle activity was determined by monitoring the flow of [3-13C]alanine into glutamate. The relative activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) is represented by the ratio of the glutamate NMR signal intensities (C2 + C3)/C4. PFDA rats show a lower (C2 + C3)/C4 glutamate ratio, suggesting greater relative activity of PDH versus PC in PFDA rats relative to controls. Differences in PDH activity may arise from differences in lipolytic activity. Our data suggest a dysfunction in fatty acid metabolism in PFDA rats and corroborate other studies which show that PFDA inhibits fatty acid oxidation.

A piezoelectric respiratory monitor for in vivo NMR.

This report describes a simple electronic device employing a piezoelectric element which serves as a sensitive detector of motion. The device is useful as a monitor of respiratory motion for nuclear magnetic resonance animal experiments in vivo. It can also provide a trigger pulse for respiratory gating experiments.

A nuclear magnetic resonance investigation of the upper airways in ferrets. I. Effects of histamine and methacholine.

Alterations induced in the upper airways of ferrets by intranasal provocation with methacholine (MC) and histamine (HS) were monitored using proton magnetic resonance imaging (MRI) and spin-spin relaxation rate (R2) measurements. Both MC and HS cause a significant increase in the MRI signal intensity and a decrease in R2 in the nasal turbinates. A dose-dependent response is observed for 20 to 315 nmol of HS, with a maximum increase in intensity of ca. 50% occurring above 80 nmol. A single unilateral challenge with MC yields a 62 +/- 3% increase in intensity. Control animals (saline-treated) show little change in image intensity. MC and HS cause decreases in the proton R2 by -27.0 +/- 5.5% and -17.2 +/- 4.3%, respectively. These data are indicative of an accumulation of fluid in the nasal airways. MRI provides an effective means to monitor changes in the nasal airways which occur as a result of pharmacological treatment.

A nuclear magnetic resonance investigation of the upper airways in ferrets. II. Contrast-enhanced imaging to distinguish vascular from other nasal fluids.

Proton magnetic resonance imaging (MRI), used in conjunction with the intravascular contrast agent albumin-(Gd-DTPA), provides a means to distinguish vascular fluids from other nasal fluids in the upper airways. Ferrets were given an intravenous dose of albumin-(Gd-DTPA) followed by an intranasal challenge with either histamine (HS) or methacholine (MC). An observed increase in image intensity indicates that HS and MC both cause an accumulation of fluids in the nasal turbinate region. The MRI data are also influenced by the presence of blood, which contains the contrast agent, and a clear distinction can be made between vascular fluids and other nasal fluids (i.e., cellular and glandular secretions). The results show that HS causes an increase in vascular fluids in the nasal turbinates while MC does not. This methodology represents a new means to investigate airway pharmacology and the pathophysiology associated with various pharmacological agents, allergens, or viral infections.

A comparative toxicological investigation of perfluorocarboxylic acids in rats by fluorine-19 NMR spectroscopy.

Male Fischer-344 rats administered a single intraperitoneal dose of perfluoro-n-octanoic acid (PFOA) or perfluoro-n-decanoic acid (PFDA) display a similar "wasting toxicity" characteristic of perfluorocarboxylic acids, with marked differences in temporal expression. Food/water consumption and urine output were monitored daily in PFOA-treated, PFDA-treated, and control rats. Fluorine-19 nuclear magnetic resonance (NMR) spectroscopy was used to monitor these fluorocarbons and possible fluoro metabolites in vivo, and to correlate differences in elimination with differences in effective toxicity. The data reveal a prolonged hypophagic response to PFDA and a more acute but transient response associated with PFOA treatment. PFOA causes a greater decline in food consumption than PFDA within the first 24 h postdose. PFOA-treated rats also show a ca. 2.5-fold increase in urine output on day 1, with only a slight increase in water consumption. In contrast to PFDA, PFOA-treated rats recover from hypophagia within 8 days. Fluorine-19 NMR spectra of various bodily fluids and liver in vivo display resonances of the parent PFOA or PFDA compounds and do not reveal any evidence of metabolism. Inorganic fluoride from dietary sources is detected in urine from both exposed and control rats. Differences in the route of excretion of PFOA vs PFDA are apparent from the spectral signal-to-noise ratio. The data suggest that PFOA is more readily excreted in the urine while PFDA is preferentially carried in bile. These apparent differences in elimination may account for their observed differences in effective toxicity. The acute transient toxicity and higher LD50 associated with PFOA may result from its rapid renal clearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of acute brain edema using quantitative magnetic resonance imaging: effects of pretreatment with dexamethasone.

We developed a quantitative magnetic resonance imaging method to permit a rapid assessment of brain water content during osmotic brain edema produced by intraperitoneal (ip) injection of distilled water. Fifteen minutes after water injection, the normalized mean image intensity (MIn) from a spin-echo pulse sequence (TE = 80 ms, TR = 1085 ms) was the same as that measured from control animals not injected with water. Sixty minutes after the water injection, the mean +/- SEM brain image MIn had increased by 10.8 +/- 2.4% compared to 3.4 +/- 0.7% in control animals (P less than 0.05). Blood plasma osmolality decreased by 6-10% during this time interval. A subsequent ip injection of hypertonic NaCl solution (100 gm/liter) caused the blood plasma osmolality and brain image MIn to return toward their initial values. MIn of cerebral gray matter correlated with tissue water content measured in parallel studies. Animals pretreated with 0.25 mg/(kg day) dexamethasone had cerebral gray matter MIn values during osmotic edema which were lower than those of untreated animals.

Relationship between total magnesium concentration and free intracellular magnesium in sheep red blood cells.

The cellular free magnesium concentration of ionophore A23187 permeabilized high potassium sheep erythrocytes was measured by 31P nuclear magnetic resonance spectroscopy, and the total cellular magnesium concentration was determined by atomic absorption spectroscopy. The free versus total cellular magnesium concentrations yield a linear relationship on a log-log scale in the concentration range from 0.3 to 1.92 mmol Mg/liter cells. Thus, free intracellular magnesium concentrations can be calculated from atomic absorption data. The method permits the estimation of physiologically or experimentally induced variations of intracellular free magnesium concentrations between 7 and 405 microM magnesium in cell water. This range encompasses the free magnesium concentration of 335 +/- 60 microM in cell water determined for untreated erythrocytes.

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study.

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.

Visibility of mammalian hepatic glycogen to the NMR experiment, in vivo.

An in vivo 13C NMR study was performed to investigate whether the relaxation properties of the C-1 carbon of the glucopyranose residues of glycogen changed as the molecule increased in size. There was no change in resonance intensity or linewidth. Therefore, no significant change in T1 or T2 was observed.