Nutritional Aspects of Breath Testing Based on Stable Isotopes

Stable isotope methodologies offer a number of possibilities for the nutritional assessment of many different processes and metabolic pathways. The application of stable isotopes has been boosted by the development of new mass spectrometers, lower costs of probes, and the risks associated with radioactive tracers. The use of ¹³C as a tracer offers all the advantages of stable isotopes and has been widely applied for measuring various types of metabolic processes. This review is focused on clinical and nutritional assessments using ¹³C breath tests.     

稳定性同位素技术对锌生物利用率的研究

锌是人体必需微量元素之一 ,锌稳态主要通过改变肠道吸收效率和内源性排泌 ,即调节生物利用率来实现。研究锌的生物利用率对全面理解锌的代谢和可靠评价人群锌营养状况起着重要的作用。稳定性同位素是目前研究矿物元素生物利用和代谢的有效工具。锌的稳定性同位素可以作为示踪物安全应用于所有人群而无放射性危害。自 80年代以来 ,许多不同的稳定性同位素方法被发展起来用以分析锌的吸收代谢 ;然而 ,一些方法学的问题尚待解决。此外 ,分析仪器的发展和应用不仅使锌稳定性同位素的应用成为可能 ,也大大降低了该技术的难度和成本。本文主要对其发展历史、应用特点、主要的研究方法及分析技术做一综述。     

Stable isotopic tracing-a way forward for nanotechnology

Numerous publications and reports have expressed health and safety concerns about the production and use of nanoparticles, especially in areas of exposure monitoring, personal use, and environmental fate and transport. We suggest that stable isotopic tracers, which have been used widely in the earth sciences and in metabolic and other health-related studies for several decades, could be used to address many of these issues. One such example we are pursuing is the use of stable isotopes to monitor dermal absorption of zinc and titanium oxides in sunscreen preparations and other personal care products. Other potential applications of this tracing approach are discussed.     

A double isotope dilution method for using stable selenium isotopes in metabolic tracer studies: analysis by gas chromatography/mass spectrometry (GC/MS)

Enriched stable isotopes of selenium are used for a double isotope dilution method employing rapid sample digestion, chelation and measurement by combined gas chromatography/mass spectrometry (GC/MS). A known quantity of an enriched selenium isotope is added as an internal standard, and samples are rapidly digested with HNO3, H3PO4 and H2O2. Undigested lipids are extracted with chloroform, and any selenate is reduced to selenite with HCl. The selenite reacts with 4-nitro-o-phenylenediamine (NPD) to form 5-nitropiazselenol (Se-NPD), which is then extracted into chloroform for subsequent GC/MS analysis. By monitoring the ion peaks in the Se-NP parent ion cluster, and by using isotope ratio measurements, it is possible in principle to use any two of the stable selenium isotopes as tracer and internal standard. The method described herein utilizes 76Se as the tracer and 82Se as the internal standard, compared to 80Se naturally present in the sample. Selenium recoveries from the digestion-chelation steps were verified by using animal tissues endogenously radiolabeled with 75Se. The method is precise, accurate, rapid and extremely specific, and should lend itself well to determining selenium in biological materials, and to following stable selenium isotopes as tracers in metabolic studies.     

Methods for assessing gastrointestinal absorption of strontium in humans by stable tracer techniques

Intestinal absorption of strontium from an oral test dose was studied in 13 healthy human volunteers using double tracer techniques with two stable strontium isotopes as tracers. Defined amounts of one isotope were administered orally, while tracer amounts of the second isotope were injected intravenously. Two different methods were used to assess the total fraction absorbed (f1 value). Fractional intestinal strontium absorption can be calculated from the ratio of the two isotopes in plasma or urine samples (in this paper called the double-isotope method) or the convolution integral technique, since both methods provide an accurate estimate of fractional absorption and yield comparable results. The latter additionally provides information on the absorption kinetics in the gastrointestinal tract. The absorption varies with respect to the chemical form and to the amount administered. Absorption patterns are characteristically different for uptake from solutions or from whole meals.     

A method for the detection and assay of iron stable isotope tracers in blood serum.

Methodology for use of stable isotopes of iron (54Fe, 57Fe, and 58Fe) as biological tracers was developed. Tracers were quantitated by measurement of ion abundances with a quadrupole mass spectrometer. The volatility of the iron was enhanced by chelation with 2,4-pentanedione prior to analysis. Samples were introduced into the mass spectrometer via a direct inlet probe. Ion abundance ratios were calculated from integrated ion current measurements obtained for selected ions in the two-ligand fragment of the chelate. Stable isotope tracer concentrations were calculated from these ratios. A prodecure was developed for the formation and purification of serum iron chelates. The method was used to analyze iron standards and blood serum samples containing known amounts of added 58Fe. The disappearance from pony serum of injected 58Fe was used as an in vivo test of the method. It was estimated that a minimum of 1.5 mg of either 54Fe, 57Fe, or 58Fe would be required to label 1 g of natural iron at detectable levels. The methods has promise as an alternative to radioisotope tracer techniques for some applications involving human subjects.     

Potential use of environmental isotopes in pollutant migration studies

This article presents the use of natural abundance stable isotope (hydrogen, carbon, nitrogen, oxygen, chlorine) analysis data as a tool for providing important information about the origin of contaminants, the contribution of different sources to a multi-source plume, characterisation of their complex transport (rate and mechanisms) and for evaluating the success of contaminated site remediation. Isotopic signatures of contaminants are useful tracers of their sources, while isotopic fractionation can be used to quantitatively assess the progress of an environmental process such as biodegradation. This new isotopic approach is reliable and can offer more information than traditional techniques in pollutant migration studies, particularly after waste disposal. During biological degradation of any organic compound, molecules containing lighter isotopes are degraded, and the portion of heavier isotopes in the substrate is increased, identifying specific microbial roles in biogeochemical cycling. Since isotopic fractionation is proportional to degradation, depending on the type of contamination, a microbial degradation of 50% to 99% of the initial concentration can be quantified using isotope ratio measurements.     

Radioisotopes for research on and control of mosquitos

Practical applications of radioactive isotopes in medicine, science, and industry have multiplied enormously during the past five years. In this paper, the author attempts to gather what is known about the use of radioactive isotopes in the research on malaria control.The development of the uranium pile for large-scale production of radioisotopes and technical progress in the making of reliable electronic equipment have greatly contributed to the application of radioactive tracers in biological research. The present knowledge of radioisotopes in mosquito and in insecticide research is discussed.     

Using stable isotopes to assess mineral absorption and utilization by children

Adequate mineral intake is a crucial part of a healthy diet for children-it supports appropriate growth and development and provides protection against childhood conditions like anemia and helps to prevent future adult diseases such as osteoporosis. Challenges in performing and interpreting studies in infants and children have hampered the accurate assessment of their mineral utilization. Many of the most powerful techniques used in adults, such as radioisotope testing, are not appropriate for use in children. In recent years, advanced mineral stable-isotope techniques have been developed to fill this gap. Pediatric applications include studies of calcium absorption and kinetics during puberty and evaluation of the calcium-iron interaction in infants and toddlers. The effects of genetics in determining calcium absorption and bone turnover may become an important research area. The goals and methods of ongoing mineral stable-isotope research in infants and children are examined in this report. In the past, the cost and difficulties in obtaining isotopes have limited such research. This situation has improved considerably, although relatively few nutrition research laboratories are prepared to perform sample analyses.     

Methodological considerations in measuring human calcium absorption: relevance to study the effects of inulin-type fructans

During the last 50 years, a variety of methods have been developed to estimate Ca absorption in man. Mass balances were initially used, but these were unable to accurately measure fractional Ca absorption because they cannot distinguish unabsorbed dietary Ca from endogenous faecal Ca excretion (excretion of previously absorbed Ca back into the gut). A number of isotopic methods have been developed that can measure true fractional Ca absorption, employing radioisotopes, stable isotopes, or both. Different methods involve collection of urine, faecal or plasma samples. Of the currently available methods, the dual isotope tracer method with a timed urine collection is probably the most precise and reliable. It is also relatively straightforward to carry out and avoids the need for a faecal collection. The purpose of the present paper is to discuss the general advantages and disadvantages of the different methods of Ca absorption. In addition, the limitations the different methods have in examining the possible effects of non-digestible oligosaccharides on Ca absorption will be discussed.     

Zinc homeostasis in man: studies using a new stable isotope-dilution technique

A new method has been developed for the study of zinc metabolism in man using the stable isotope 67Zn. The technique involves intravenous infusion of the isotope followed by measurements of the plasma and faecal enrichments over a period of days. A procedure for the analysis of Zn isotopes in plasma and faeces is described which requires the separation of Zn from other elements using the chelator dithizone before analysis by thermal-ionization mass spectrometry. The stable isotope technique has been used in conjunction with a metabolic balance study to obtain measurements of Zn absorption and gastrointestinal secretion in a normal subject. Preliminary measurements of the size of the exchangeable pool of Zn have been made as have estimates of the rates of plasma and whole-body Zn turnover. Following an increase in dietary Zn the body appeared to respond in two ways. The gastrointestinal secretion of Zn increased immediately, but only by a relatively small amount. The absorption of Zn initially increased in proportion to the increase in dietary levels but then decreased within 4 d by an amount sufficient to restore Zn balance.     

Measuring very low density lipoprotein-triglyceride kinetics in man in vivo: how different the various methods really are

PURPOSE OF REVIEW: The purpose of this article is to briefly outline the methods that are currently available for the determination of very low density lipoprotein-triglyceride (VLDL-TG) kinetics in man in vivo. RECENT FINDINGS: A number of novel methodologies have been developed over the years for quantifying VLDL-TG production, clearance, and turnover rates. Besides the splanchnic arteriovenous balance technique, tracer methods with radioactive and, more recently, stable isotopes have been widely used. Most of the latter approaches utilize an isotopically labelled substrate, such as glycerol, fatty acid or acetate, which is eventually incorporated into a VLDL-TG moiety, and monitor the time course of change in specific activity or enrichment. A procedure of in vivo labelling of VLDL-TG with stable isotopes and use of the labelled VLDL-TG as a tracer has also been described in man. There is, however, considerable variability in estimates of VLDL-TG kinetics obtained by the various techniques, which cannot be readily attributed to normal physiological variation. Still, a large part of this discrepancy may be related to differences in VLDL-TG pool size within the normal range, which seem to account for approximately 40-50% of the total variance in VLDL-TG kinetics in both men and women. SUMMARY: Several methods are available for quantifying VLDL-TG kinetics in man in vivo, varying in the selection of tracer, mode of administration and sampling, and data analysis. These inherent features, along with different pool sizes, result in multifold variable estimations of VLDL-TG kinetic parameters.     

Measuring calcium absorption and utilization in humans

PURPOSE OF REVIEW: Calcium metabolism is comprised primarily of absorption, urinary excretion, endogenous secretion and bone turnover. This review evaluates recent findings relating to the role of genetic and environmental factors, especially diet, on perturbing parameters of calcium metabolism. Calcium dynamics are studied with the use of isotopic tracers. We also cover state-of-the-art methods for stable calcium isotope ratio analysis and offer insights on experimental design. RECENT FINDINGS: Some progress has been made identifying genetic and hormonal regulators of calcium absorption. Much progress has been made in understanding the role of diet on influencing calcium retention, especially with regard to dietary protein and salt. Long-held views on dietary factors thought to contribute to bone loss through urinary calcium loss have been shown to have no impact on net calcium retention because of compensatory changes in other aspects of calcium metabolism. SUMMARY: Much more work needs to be done on understanding genetic regulators of calcium metabolism. Despite recent advances in our knowledge of dietary influences on calcium metabolism, more studies are needed on the role of environmental factors, especially physical activity.     

Use of stable isotopes to study carbohydrate and fat metabolism at the whole-body level

The present review discusses the advantages and limitations of using stable-isotope tracers to assess carbohydrate and fat metabolism at the whole-body level. One advantage of stable- (v. radioactive-) isotope tracers is the relative ease with which the location of a label within a molecule can be determined using selected-ion-monitoring GC-mass spectrometry (SIM-GC-MS). This technique minimizes potential problems due to label recycling, allows the use of multiple-labelled compounds simultaneously (e.g. to quantify glucose cycling), and perhaps most importantly, has led to the development of unique stable-isotope methods for, for example, quantifying gluconeogenesis. However, the limited sensitivity of SIM-GC-MS sometimes requires that relatively large amounts of a stable-isotope tracer be used, thus increasing cost and potentially altering metabolism. At least theoretically, stable- (or radioactive-) isotope tracers can also be used in conjunction with indirect calorimetry to estimate utilization of muscle glycogen or triacylglycerol stores, thus potentially circumventing the need to obtain muscle biopsies. These calculations, however, require certain critical assumptions, which if incorrect could lead to major errors in the values obtained. Despite such limitations, stable-isotope tracers provide a powerful and sometimes unique tool for investigating carbohydrate and fat metabolism at the whole-body level. With continuing advances in availability, instrumentation and methods, it is likely that stable-isotope tracers will become increasingly important in the immediate future.     

Use of bioelectrical impedance techniques for monitoring nutritional status in patients on maintenance dialysis.

Malnutrition continues to be an important correlate of survival in dialysis patients. Nutritional surveillance at the clinical level requires use of simple, reasonably accurate, and easily accessible techniques for multicompartmental body composition analysis. Unfortunately, although gold standard methodologies (body density by underwater weight, total body water by isotope dilution, bone mineral content by neutron activation, total body potassium by (40)K whole body gamma counting) provide very precise assessments, they are not applicable to routine clinical practice. Because of its availability and simplicity, bioelectrical impedance (BEI) has significant potential as a complement to standard anthropometric techniques in the nutritional monitoring of patients with chronic renal failure. Consistency of technique and standardization of BEI equipment are essential for reproducibility of results. Several studies have validated the use of total body water by BEI as a surrogate for isotope dilution methods in dialysis patients, whereas others have established an excellent correlation with the volume of distribution of urea as measured by urea kinetic volume. Bioimpedance analysis for measurement of lean body mass has been extensively evaluated in stable healthy populations, with results similar to those obtained using hydrodensitometry and total body potassium. In dialysis patients, accuracy is contingent on a stable hydration status and/or appropriate correction for changes in extracellular volume status over time. Recent publication of bioimpedance norms for the hemodialysis population allows better comparisons with the national reference population studied as part of the National Health and Nutrition Examination Survey III (US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics, Hyattsville, MD). BEI methodology is a practical bedside tool for assessment of body composition that provides more consistent and reproducible results than standard anthropometry alone.     

稳定同位素在蛋白质合成率测定中的应用

由于放射性同位素对人体有害,故逐渐被稳定同位素取代,同时随着质谱仪的出现和发展,稳定同位素的应用日趋广泛,目前最常用的稳定同位素测定蛋白质合成率方法有两种:即持续灌注法及冲击法.     

Les traceurs isotopiques stables en nutrition humaine : que peut-on faire avec ?

This review describes many aspects of the use of stable isotopes tracers for the study of human metabolism. These molecules are inoffensive and bring informations about plasma substrates fluxes and synthesis of complex molecules (proteins, triglycerides, glycogen). They can be associated together and to other techniques such as clamp or indirect calorimetry during the same experiment in the same subject. They help to better understand physiological and pathophysiological mecanisms sustaining the maintenance of a good or an altered nutritional status. In the field of health claims, they are useful to bring the demonstration of the the metabolic fate of novel foods or of their benefits for health.     

An in vitro system for measuring intrinsic dietary mineral exchangeability: alternative to intrinsic isotopic labeling

The primary purpose for the in vitro system described here was to provide an alternative method to intrinsic isotopic labeling for determining exchangeability of intrinsic food mineral with extrinsic inorganic mineral or one of its isotopes. In this system, foods or food mixtures extrinsically tagged with an isotope of the mineral of interest are incubated successively in media simulating peptic or pancreatic digestion. Radioactivity and mineral measurements are made on a)the mixture before digestion and after b)peptic and c)successive peptic pancreatic (P-Pa) digestion. Exchangeability is determined by comparing the specific activity in the peptic and P-Pa digest supernatants to that in the mixture before digestion. In addition, determination of the total soluble mineral after P-Pa digestion provides a measurement of potential bioavailability. The procedure was tested for calcium (Ca) exchangeability and bioavailability from a number of foods and food combinations with 45Ca as the tracer. It could be used for any mineral, however, including non-nutrient or toxic elements. It is likely to be most useful in association with human studies carried out with extrinsic stable (non-radioactive) or short-lived radioactive isotopes that are unsuitable for intrinsic labeling.     

Vitamin A: biomarkers of nutrition for development

Vitamin A is essential for multiple functions in mammals. Without vitamin A, mammals cannot grow, reproduce, or fight off disease. Because of its numerous functions in humans, biomarkers of vitamin A status are quite diverse. Assessment of liver reserves of vitamin A is considered the gold standard because the liver is the major storage organ. However, this measure is not feasible in human studies. Alternative biomarkers of status can be classified as biological, functional, histologic, and biochemical. Historically, signs of xerophthalmia were used to determine vitamin A deficiency. Before overt clinical damage to the eye, individuals who suffer from vitamin A deficiency are plagued by night blindness and longer vision-restoration times. These types of assessments require large population-based evaluations. Therefore, surrogate biochemical measures of vitamin A status, as defined by liver reserves, have been developed. Serum retinol concentrations are a common method used to evaluate vitamin A deficiency. Serum retinol concentrations are homeostatically controlled until liver reserves are dangerously low. Therefore, other biochemical methods that respond to liver reserves in the marginal category were developed. These included dose-response tests and isotope dilution assays. Dose-response tests work on the principle that apo-retinol-binding protein builds up in the liver as liver reserves become depleted. A challenge dose of vitamin A binds to this protein, and serum concentrations increase within a few hours if liver vitamin A concentrations are low. Isotope dilution assays use stable isotopes as tracers of total body reserves of vitamin A and evaluate a wide range of liver reserves. Resources available and study objectives often dictate the choice of a biomarker.     

An introduction to the use of tracers in nutrition and metabolism

The present article is a review written at a level suitable for students and new workers to the field of techniques in common current use for the measurement of static and dynamic features of metabolism, especially nutritional metabolism. It covers the nature of radioactive and stable-isotope tracers, the means of measuring them, and the advantages and disadvantages of their use. The greater part of the review deals with methods for the measurement of pool sizes and metabolic processes, with the emphasis being on protein metabolism, a field the author knows best. The examples given are from a variety of sources, including the work of the author, but the principles underlying the techniques are universally applicable to all metabolic investigations using tracers.