A novel extrinsic reference method for assessing the vitamin A value of plant foods.

BACKGROUND: The amounts of vitamin A that are metabolically derived from specific carotene-containing foods are largely unknown. OBJECTIVE: We sought to develop an improved method for estimating the metabolic vitamin A potential of provitamin A carotenoids by using [2H4]retinyl acetate (d4-RA) as an extrinsic reference standard. DESIGN: Healthy subjects consumed a standardized test meal containing 6 mg beta-carotene as either raw carrot or spinach, either 20 or 1 g added fat, and 6.0 micromol d4-RA. Concentrations of unlabeled (d0) retinyl esters (RE), labeled (d4) RE, and carotenoids in the plasma triacylglycerol-rich lipoprotein fraction (d < 1.006 kg/L) were determined in serial blood samples with HPLC and gas chromatography-mass spectrometry. Baseline-corrected areas under the curve for d0-RE, d4-RE, and carotenoids were calculated, and the masses of absorbed d0-retinol and carotenes were estimated assuming 80% absorption of the d4-RA reference dose. RESULTS: In trials with ample (20 g) fat (n = 6), 7 +/- 4% of the 6 mg beta-carotene ingested was taken up as beta-carotene plus RE with 0.3 +/- 0.1 mg as retinol. Test meals without carotenes yielded no beta-carotene or d0-RE response and there was no effect of treatment (either fat amount or vegetable, n = 6) on the mean d4-RE area under the curve. The lower-than-expected vitamin A yields were attributed to poor intestinal uptake rather than to low conversion of beta-carotene to RE. CONCLUSION: The triacylglycerol-rich lipoprotein and d4-RA method, which controls for variation in chylomicron kinetics in vivo and RE recovery during analysis, is useful for obtaining quantitative estimates of the vitamin A potential of single meals.

Real time biodetection of individual pathogenic microorganisms in food and water.

The primary objective of this research is to examine the feasibility of using an innovative technique based on laser-induced fluorescence coupled with flow cytometry to detect pathogenic microorganisms in food or water in real time. Our initial application is the rapid detection of E. coli O157:H7 in ground beef. The research performed demonstrated conclusively that this approach is feasible, and that the technique has key advantages over current alternatives including: it is (1) able to totally examine a large volume of food or water in real time, (2) capable of detecting single microorganisms (alternative techniques require in excess of 10(4) microorganisms), (3) intrinsically automatic, and (4) sensitive only to the selected bacteria. We have demonstrated the feasibility of detecting individual E. coli bacteria with a breadboard system. The performance of this system allows for rapid detection of individual specific pathogenic microorganisms. Two of the most significant commercial applications of this technique are the detection of infectious microorganisms in contaminated food and water. Food-borne microbial pathogens account for approximately 7 million illnesses and 9,000 deaths in the U.S. annually, with an estimated economic loss of at least $6 billion [1]. In addition, this method has the potential for a broad range of other commercial applications, including the detection of small numbers of molecules, such as the ultrasensitive detection of explosives and groundwater contaminants.

Quantum-cascade laser measurements of stratospheric methane and nitrous oxide.

A tunable quantum-cascade (QC) laser has been flown on NASA's ER-2 high-altitude aircraft to produce the first atmospheric gas measurements with this newly invented device, an important milestone in the QC laser's future planetary, industrial, and commercial applications. Using a cryogenically cooled QC laser during a series of 20 aircraft flights beginning in September 1999 and extending through March 2000, we took measurements of methane (CH(4)) and nitrous oxide (N(2)O) gas up to ~20 km in the stratosphere over North America, Scandinavia, and Russia. The QC laser operating near an 8-mum wavelength was produced by the groups of Capasso and Cho of Bell Laboratories, Lucent Technologies, where QC lasers were invented in 1994. Compared with its companion lead salt diode lasers that were also flown on these flights, the single-mode QC laser cooled to 82 K and produced higher output power (10 mW), narrower laser linewidth (17 MHz), increased measurement precision (a factor of 3), and better spectral stability (~0.1 cm(-1) K). The sensitivity of the QC laser channel was estimated to correspond to a minimum-detectable mixing ratio for methane of approximately 2 parts per billion by volume.

Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin.

Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specific cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 microM), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of gamma- and alpha-tocopherol in rat primary hepatocytes, and metabolism of gamma- and delta-tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 microM. These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the first evidence of a specific enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism.

Chronic treatment with phentermine combined with fenfluramine lowers plasma serotonin.

As expected on the basis of published research in both humans and animals, treatment with phentermine/fenfluramine lowers plasma 5-hydroxytryptamine [corrected], whereas treatment with phentermine had no significant effect. In light of these findings, future research should focus on mechanisms other than increased plasma 5-hydroxytryptamine [corrected] to explain how fenfluramine increases the risk of primary pulmonary hypertension and valvular heart disease.

A novel 5'-carboxychroman metabolite of gamma-tocopherol secreted by HepG2 cells and excreted in human urine.

HepG2 cells were incubated with a medium containing fetal bovine serum enriched with RRR-gamma-tocopherol (gamma-TOH). After 48 h the medium was extracted and analyzed for gamma-TOH metabolites by gas chromatography-mass spectrometry. In addition to gamma-CEHC, the 3'-carboxychroman metabolite of gamma-TOH previously reported in human urine, these cells secreted a second substance whose extraction and mass spectral characteristics were consistent with those of the 5'-carboxychroman analog of gamma-CEHC, 2,7, 8-trimethyl-2-(delta-carboxymethylbutyl)-6-hydroxychroman. This is the first report of metabolism of gamma-TOH to carboxychroman metabolites in cell culture. Analysis of human urine samples revealed the consistent presence of the novel 5'-carboxychroman metabolite, along with that of gamma-CEHC. Oral supplementation with purified RRR-gamma-TOH resulted in elevated urinary concentrations of both metabolites, although the concentration of the 5'-gamma-carboxychroman metabolite was consistently and substantially less than that of gamma-CEHC. The presence of both metabolites is consistent with the involvement of an omega-oxidation-like process in the phytyl tail shortening of gamma-TOH to water soluble metabolites excreted in urine.

Pharmacologic induction of weight loss to treat type 2 diabetes.

OBJECTIVE: Most individuals with type 2 diabetes are overweight, and weight loss for them is an important therapeutic objective. However, usual weight-loss strategies have generally not produced sustained weight loss. Pharmacologic agents to assist weight loss might be useful, but no long-term data on their effectiveness and safety in patients with type 2 diabetes are available. We therefore initiated a 2-year placebo-controlled trial of the weight-loss medications fenfluramine and phentermine in type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: A total of 44 overweight (> 120% ideal body weight) subjects with type 2 diabetes were enrolled in a randomized, placebo-controlled, double-blind trial of fenfluramine and phentermine. All subjects received intensive nutrition counseling, an exercise prescription, and instruction in behavior modification. Subjects were randomly assigned to 20 mg fenfluramine three times a day and 37.5 mg phentermine daily (n = 23) or dual placebos (n = 21). Diabetes medications were adjusted as necessary to achieve glycemic goals. Changes in weight, glycemia, lipemia, and blood pressure were assessed every 2 months, as were adverse events. In September 1997, when fenfluramine was withdrawn from the U.S. market, fenfluramine was stopped in all subjects. Thus the length of drug treatment varied, but 16 subjects (8 in each group) reached 12 months of treatment. Only data obtained before the withdrawal of fenfluramine are included in this report. RESULTS: A study termination, diabetes medications had been reduced in 1 subject in the placebo group (5%) and 11 subjects in the drug treatment group (52%) (P = 0.005). Drug treatment resulted in significant reductions in body weight, BMI, and HbA1c at all time points through 6 months. Changes in weight at 6 months were -2.7 +/- 1.4 kg (mean +/- SEM) with placebo treatment and -9.6 +/- 1.5 kg with drug treatment (P = 0.003). Even though more subjects in the drug treatment group required reductions in diabetes medications, at 6 months, changes in HbA1c were -0.3 +/- 0.2% with placebo treatment and -1.6 +/- 0.3% with drug treatment (P = 0.002). Fasting plasma glucose and triglycerides were significantly reduced at some time points with drug treatment. No serious adverse events attributable to study medications were observed. CONCLUSIONS: Premature study termination decreased the power of our study at later time points. However, our data suggest that weight loss medications are an effective treatment for type 2 diabetes during active weight loss. Whether the benefit persists after weight loss has stopped remains to be determined.

Bioavailability of carotenoids in human subjects.

There is growing need for accurate information regarding the bioavailability of carotenoids, both with respect to carotenoids per se and to the vitamin A value of provitamin A carotenoids in foods or supplement preparations. Little quantitative information is currently available, owing primarily to the lack of adequate methods to assess carotenoid bioavailability. Methods applied to xenobiotic drugs are in most cases not useful for carotenoids, many of which circulate in appreciable quantities in human plasma. Reported ranges of carotenoid bioavailability (% dose absorbed) range from 1-99, and variability is generally high both within and between treatments. With the current methods, relative bioavailability is more readily assessed than absolute bioavailability. The most commonly applied methods include measuring the increase in plasma carotenoid concentration following chronic intervention, and use of postprandial chylomicron (PPC) carotenoid or retinyl ester response following a single dose of carotenoid. The advantages and limitations of these approaches, together with examples of each, are discussed. A new PPC approach utilizing extrinsic-stable-isotope-labelled vitamin A (2H4-labelled retinyl acetate) is under development in our laboratory, and examples of its application are presented. The currently available data suggest that oil solutions of carotenoids are more bioavailable than those from food matrices, and heating can improve the bioavailability of carotenoids from some food products. Increased availability of labelled carotenoids and retinoids should aid the development of reliable methods of carotenoid bioavailability assessment. Such data are needed for dietary recommendations, supplement formulation, and design of intervention strategies involving carotenoids.

Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans.

Little is known of the post-absorptive, metabolic fate of gamma-tocopherol, the major form of vitamin E in North American diets. The objective of this study was to determine the extent of urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a recently identified metabolite of gamma-tocopherol. A method for measurement of urinary gamma-CEHC was developed, using gas chromatography-mass spectrometry (GC-MS) with a deuterated internal standard, 2,7,8-trimethyl-2-(beta-carboxyethyl)-(3, 4-2H2)-6-hydroxychroman (d2-gamma-CEHC). This standard was synthesized by dehydrogenation of 6-acetyl-gamma-CEHC followed by deuteration of the resulting 3,4-double bond. The use of d2-gamma-CEHC resulted in accurate determinations of the concentration of d0-gamma-CEHC in human urine. Urine samples containing added d2-gamma-CEHC were treated with beta-glucuronidase, extracted with an organic solvent, and analyzed by GC-MS. Analysis of 24-h urine pools from healthy subjects revealed gamma-CEHC concentrations, normalized against creatinine, ranging from 2.5 to 31.5 micromol/g creatinine, or a total of 4.6 to 29.8 micromol per day. These results correspond to 2-12 mg gamma-tocopherol excreted daily as gamma-CEHC in the urine. Given an estimated mean intake of gamma-tocopherol of 20 mg/day, catabolism of gamma-tocopherol to gamma-CEHC, followed by glucuronide conjugation and urinary excretion, is a major pathway for elimination of gamma-tocopherol in humans.

Effect of UV exposure and beta-carotene supplementation on delayed-type hypersensitivity response in healthy older men.

OBJECTIVES: The aims of this study were to determine if ultraviolet light (UV) is immunosuppressive in healthy older males, if beta-carotene (betaC) supplementation could prevent any observed UV-induced immunosuppression, and to compare these effects with those observed previously in younger men. METHODS: The study was a placebo-controlled, randomized trial that employed a 2 x 2 factorial design. Healthy older men (mean age 65.5 years) received 30 mg betaC or placebo daily throughout the 47-day trial, while on a low carotenoid diet. After 28 days, half of each group received 12 suberythemic exposures to UV over a 16-day period. Delayed-type hypersensitivity (DTH) tests and plasma carotenoid assays were performed at baseline, pre-UV and post-UV time points, with DTH testing performed on an area of skin protected from UV exposure. RESULTS: UV exposure resulted in significantly suppressed DTH response in the placebo group but not in the betaC-UV group. While there was no significant interaction between betaC supplementation and UV on DTH response, there was a significant inverse relationship between final plasma betaC concentration and extent of UV-induced suppression of DTH response. A similar correlation existed among subjects not exposed to UV. CONCLUSIONS: Suberythemic UV exposure was immunosuppressive, as measured by DTH response, in healthy older men as in younger men. Higher plasma betaC was significantly associated with maintenance of DTH response, although the extent of protective effect of betaC appeared less than previously observed in younger subjects. The attenuated effect of betaC in the older UV-exposed subjects may have resulted in part from muted plasma betaC responses to betaC supplementation and/or higher plasma vitamin E levels than those of younger men. The finding that stronger DTH responses were associated with higher plasma betaC concentrations in both UV and non-UV subjects further supports a role for this nutrient in immunomodulation.

Evidence of a role for fat-free body mass in modulation of plasma carotenoid concentrations in older men: studies with hydrodensitometry.

We examined the relationship between body composition and changes in plasma carotenoid concentration in response to dietary carotenoid restriction or beta-carotene (betaC) supplementation in healthy older men. Subjects (mean age 65 y) were assigned randomly to supplement (30 mg betaC/d) or placebo groups, and all subjects consumed a standard low carotenoid basal diet plus 1.5 mg betaC/d as carrots. Body composition was measured at baseline by hydrodensitometry, and plasma carotenoids were measured at baseline and after 28 d of treatment by HPLC. Baseline plasma total carotenoid concentration was significantly and negatively correlated with body mass index (BMI) and fat-free mass (FFM) but not with fat mass, whereas baseline betaC concentration was negatively associated with all three variables. The increase in plasma betaC concentration in response to betaC supplementation was significantly and inversely correlated with BMI and FFM but not with fat mass. Likewise, the decline in plasma total carotenoid concentration in the placebo group was also significantly and inversely related to BMI and FFM but not to fat mass. Thus, FFM seems to be an important determinant of plasma carotenoid concentrations and to explain a substantial portion of the often-observed relationship between BMI and blood carotenoid levels. Fat-free mass seems to represent a dynamic reservoir that dampens short-term changes in plasma carotenoid concentrations during fluctuation in carotenoid intake.

Evidence of cis-trans isomerization of 9-cis-beta-carotene during absorption in humans.

Absorption and metabolism of [13C]9-cis-beta-carotene ([13C]9c beta C) was studied in three subjects after a single oral dose. Subjects given 1.0 mg [13C]beta-carotene (mean: 99.4% 9-cis-beta-carotene, 0.6% all-trans-beta-carotene; dose A) had substantial concentrations of [13C]all-trans-beta-carotene ([13C]tr beta C) and [13C]all-trans retinol ([13C]retinol) but very low concentrations of [13C]cis-beta-carotene ([13C]cis beta C) in saponified plasma 5 h after dosing, as determined by HPLC and isotope-ratio mass spectrometry. There was no evidence of appreciable absorption of [13C]9-cis retinol. To determine the proportion of [13C]tr beta C and [13C]retinol derived from [13C]9c beta C, a second set of studies in the same subjects was performed with the same isomeric composition except with 13C labeling only in all-trans-beta-carotene (dose B). The results indicated that > 95% of plasma [13C]tr beta C and [13C]retinol observed after dose A was derived from [13C]9c beta C. The concentrations of [13C]tr beta C observed, in excess of that derived from the trace amounts of [13C]tr beta C in the dose, indicated that a significant proportion of the [13C]9c beta C dose was isomerized to [13C]tr beta C before entering the bloodstream. Although precise quantitative estimates of the extent of isomerization of 9-cis-beta-carotene could not be made, it is apparent that cis-trans isomerization of 9-cis-beta-carotene to all-trans-beta-carotene contributed to the near absence of postprandial plasma 9-cis-beta-carotene after its oral administration in humans. The observation of different ratios of beta-carotene to retinol between the two dosing protocols suggests that isomerization did not occur exclusively before uptake by the intestinal mucosa. These results indicate that isomerization of ingested 9-cis-beta-carotene before its secretion into the bloodstream limits the potential supply of 9-cis retinoids to tissues, and increases the vitamin A value of 9-cis-beta-carotene.

Metabolic effects of dietary sucrose in type II diabetic subjects.

OBJECTIVE: To assess in diabetic subjects the effects of dietary sucrose on glycemia and lipemia. RESEARCH DESIGN AND METHODS: Twelve type II diabetic subjects consumed, in random order, two isocaloric, 55% carbohydrate study diets for 28 days. In one diet, 19% of energy was derived from sucrose. In the other diet, < 3% of energy was derived from sucrose, and carbohydrate energy came primarily from starch. Both study diets were composed of common foods. All meals were prepared in a metabolic kitchen where foods were weighed during meal preparation. RESULTS: No significant differences were noted between the study diets at any time point in mean plasma glucose. At day 28, mean plasma glucose values for the sucrose diet were 9.6 +/- 0.5 mM and for the starch diet were 9.4 +/- 0.6 mM (P = 0.63). Also, no significant differences were observed between the study diets in urine glucose, fasting serum total, HDL, or LDL cholesterol; fasting serum TG; or peak postprandial serum TG. CONCLUSIONS: A high sucrose diet did not adversely affect glycemia or lipemia in type II diabetic subjects.

Nonideal mixing of phosphatidylserine and phosphatidylcholine in the fluid lamellar phase.

The mixing of phosphatidylserine (PS) and phosphatidylcholine (PC) in fluid bilayer model membranes was studied by measuring binding of aqueous Ca2+ ions. The measured [Ca2+]aq was used to derive the activity coefficient for PS, gamma PS, in the lipid mixture. For (16:0, 18:1) PS in binary mixtures with either (16:0, 18:1)PC, (14:1, 14:1)PC, or (18:1, 18:1)PC, gamma PS > 1; i.e., mixing is nonideal, with PS and PC clustered rather than randomly distributed, despite the electrostatic repulsion between PS headgroups. To understand better this mixing behavior, Monte Carlo simulations of the PS/PC distributions were performed, using Kawasaki relaxation. The excess energy was divided into an electrostatic term Uel and one adjustable term including all other nonideal energy contributions, delta Em. Uel was calculated using a discrete charge theory. Kirkwood's coupling parameter method was used to calculate the excess free energy of mixing, delta GEmix, hence In gamma PS,calc. The values of In gamma PS,calc were equalized by adjusting delta Em in order to find the simulated PS/PC distribution that corresponded to the experimental results. We were thus able to compare the smeared charge calculation of [Ca2+]surf with a calculation ("masked evaluation method") that recognized clustering of the negatively charged PS: clustering was found to have a modest effect on [Ca2+]surf, relative to the smeared charge model. Even though both PS and PC tend to cluster, the long-range nature of the electrostatic repulsion reduces the extent of PS clustering at low PS mole fraction compared to PC clustering at an equivalent low PC mole fraction.

Metabolic effects of dietary fructose in diabetic subjects.

OBJECTIVE: To assess the metabolic effects of chronic dietary fructose consumption in diabetic subjects. RESEARCH DESIGN AND METHODS: Six type I and 12 type II diabetic subjects consumed, in random order, two isocaloric study diets for 28 days. In one diet, 20% of energy was derived from fructose. In the other diet, < 3% of energy came from fructose, and carbohydrate energy was derived primarily from starch. Both study diets were composed of common foods. All meals were prepared in a metabolic kitchen where all foods were weighed during meal preparation. RESULTS: Mean plasma glucose, urine glucose, and serum glycosylated albumin values were lower during the fructose diet than during the starch diet, but the differences achieved only marginal statistical significance. The day-28 value for mean plasma glucose was 12.5% lower (P = 0.03) during the fructose diet than during the starch diet. At days 14, 21, and 28, fasting serum cholesterol and LDL cholesterol were both significantly higher during the fructose diet than during the starch diet. The day-28 values for serum cholesterol and LDL cholesterol during the fructose diet were 6.9% (P = 0.008) and 10.9% (P = 0.002) higher, respectively, than the corresponding values during the starch diet. No differences were observed between the study diets in fasting serum HDL cholesterol, fasting serum triglycerides, peak postprandial serum triglycerides, or fasting serum lactate. Peak postprandial serum lactate was significantly higher during the fructose diet. Type I and type II diabetic subjects responded to the diets in a consistent way, but type I subjects experienced significantly more hypoglycemia during the fructose diet than during the starch diet. CONCLUSIONS: A high-fructose diet may result in reduced glycemia in diabetic subjects but at the expense of increased fasting serum total and LDL cholesterol.

Metabolic effects of dietary fructose in healthy subjects.

To determine if dietary fructose causes adverse metabolic effects, we used a crossover design to compare a diet containing 20% of energy from fructose with an isoenergic high-starch diet that contained less than 3% fructose. Fourteen healthy subjects consumed each diet for 28 d. There were no significant differences between the diets in the mean values of hemoglobin A1C, serum glycosylated albumin, fasting plasma glucose, peak postprandial plasma glucose, integrated plasma glucose, fasting serum lactate, or fasting serum triglycerides. Peak postprandial serum lactate was significantly higher during the fructose diet at days 1, 7, and 14 but not at days 21 or 28. Peak postprandial serum triglycerides were significantly higher only at day 1 of the fructose diet. Day-28 fasting serum total and LDL cholesterol for the fructose diet were 9.0% and 11.0% higher, respectively, than the corresponding values for the starch diet. A high-fructose diet compared with a high-starch diet resulted in significantly higher fasting serum total and LDL cholesterol and also caused transient changes in postprandial serum lactate and triglycerides.

Thermodynamics of mixing of phosphatidylserine/phosphatidylcholine from measurements of high-affinity calcium binding.

A simple model system is described that allows measurement of equilibrium Ca2+ binding to multilamellar vesicle mixtures of palmitoyloleoylphosphatidylserine (P,O-PS) and dimyristoleoylphosphatidylcholine (MO,MO-PC). The constraint of the chemical equilibrium among aqueous Ca2+, hydrated P,O-PS/MO,MO-PC, and Ca(PS)2, together with measurements of the Ca2+ concentration in equilibrium with defined PS/PC ratios, enables the determination of the thermodynamic activity of the lipids. The activity coefficient of dilute P,O-PS in PC is analyzed in terms of the partial molal free energy to transfer P,O-PS from an environment of PS to an environment of PC. This study of P,O-PS/MO,MO-PC, by comparison with the earlier study of P,O-PS/P,O-PC [Feigensen, G.W. (1989) Biochemistry 20, 1270-1278], reveals that the excess partial molal free energy to transfer P,O-PS from P,O-PS to P,O-PC is -0.7 kcal mol-1. This free energy change arises in part from the favorable transfer of the negatively charged phosphoserine headgroup from an environment of negative charges to an environment of zwitterions. The contribution of acyl chain mismatch to the partial molal free energy to transfer P,O-PS from P,O-PS to MO,MO-PC is found to be approximately +0.7 kcal mol-1. This value is much larger than that of the excess partial molal free energy of mixing in isotropic solutions of linear hydrocarbons that differ in chain length or unsaturation.