Isotope Dilution Research Articles

Investigation of bisphenol S (BPS) in packaged fish, meat, cheese, and price labels on their corresponding packages

As an alternative to bisphenol A (BPA), bisphenol S (BPS) has been used as an ink developer in thermal paper products including price labels on food packaging which have been suggested as the sources of BPS found at high levels in packaged fish samples. BPS in the printed price labels glued onto the outside of plastic film could migrate indirectly from the printed surface through the paper, adhesive and film into the food. In order to investigate if price labels could also be the sources of BPS detected in the meat samples in our previous studies, meat and other food samples packaged under different conditions were collected, and BPS in these samples together with the price labels on the corresponding packaging were extracted with solvent followed by solid phase extraction and stable isotope dilution LC-MS/MS analysis. BPS was detected at very high levels (161.7–222.4 µg/cm2) in all the five sticker type of price labels, indicating BPS being the dominant if not the sole ink developer. BPS was also detected in all the 26 continuous roll type of price labels but at very low levels (0.017–18 ng/cm2), indicating that the dominant ink developer is likely one of the other alternatives, rather than BPS. Despite BPS being detected in all price labels on packaging of fish, meat, and cheese samples, BPS was not detected or detected in only a few fish, meat, and cheese samples at levels considerably lower than the current EU specific migration limit (SML) of 50 ng/g food for BPS authorised under Regulation (EU) 10/2011.

Use of the Paired Retinol Isotope Dilution Test, but with a Single Isotope Dose, to Assess the Impact of a Vitamin A Intervention on Vitamin A Stores in Theoretical Children with Low Stores

BackgroundAs currently applied, the paired retinol isotope dilution (RID) test, which is used to assess the impact of a vitamin A intervention on vitamin A total body stores (TBS), requires 2 doses of stable isotope-labeled vitamin A. ObjectivesThe objectives of this study were to evaluate use of a single isotope dose (4 μmol) to assess TBS by RID before and after intervention in theoretical children with low/moderate TBS. MethodsWe selected 6 theoretical children with assigned values for TBS ranging from 82 to 281 μmol. Using Simulation, Analysis and Modeling software, we simulated the variable [plasma retinol specific activity (SAp)] and coefficients (Fa and S) used in the RID equation TBS (μmol) = FaS × 1/SAp in both the unsupplemented steady state at day 14 postdosing and during the subsequent 4 mo without or with vitamin A supplementation [2.8 μmol retinol/d (801 μg retinol activity equivalents/d)]. ResultsFraction of dose in plasma on day 150 compared with day 14 was similar in the unsupplemented and supplemented conditions [geometric mean, 32% (range, 20%–48%) and 30% (20%–48%), respectively] and simulated values for FaS were similar under the 2 conditions. After 2 and 4 mo of daily vitamin A supplementation with 2.8 μmol/d, TBS was 78% and 128% higher, respectively, than without supplementation. ConclusionsResults indicate that the paired RID method can successfully be done using a single 4 μmol dose of stable isotope. Furthermore, because values for the RID coefficient FaS were similar in the unsupplemented and vitamin A-supplemented conditions, these results in theoretical children indicate that FaS determined by population (“super-subject”) modeling of steady state vitamin A kinetic data could be used to predict TBS by RID after a vitamin A intervention in individuals from the same or a similar group.

Nasal exudate for diagnosis of stroke: fundamental studies through iron fractionation, total iron, and targeted protein determinations

During the last years, there has been an increasing research interest in the analysis of biological fluids requiring non-invasive sampling for biomedical and clinical applications. In this work, we have focused on the nasal exudate with the aim of investigating the potential use of this fluid to know the role of iron in stroke and also for diagnosis. Potential differences in the nasal exudate, collected in swabs, from diagnosed hemorrhagic stroke, ischemic stroke, and control groups were investigated with regard to total iron by inductively coupled plasma-mass spectrometry, iron fractionation studies by size exclusion chromatography together with post-column isotope dilution analysis, and four proteins containing iron (ferritin, transferrin, lactoferrin, and ferroportin) with ELISA kits. All these analyses represent an analytical challenge, considering the rather limited amount of sample (10–40 mg) available, being the nasal exudate extracted from the swab with 300 µL 10 mM Tris/HCl, pH = 7.4. Studies to obtain reliable analytical information, such as the blank contribution of the sampling step, evaluation of the extraction efficiency of the nasal exudate from the swab, and normalization strategies for data treatment, have been carried out. Results showed that despite the limited number of investigated samples, fractionation studies as well as the concentrations of ferritin and ferroportin obtained with ELISA kits showed a differential behavior between the different cohorts.

Molecular Aroma Composition of Vanilla Beans from Different Origins.

The demand for natural Vanilla has increased rapidly, creating the need for more potential sources of high-quality Vanilla essence. Understanding the geographical influences on the aroma profile of Vanilla is essential. This study demonstrates the first comparative analysis of odorant compositions in the three most important Vanilla varieties: Vanilla planifolia, Vanilla pompona, and Vanilla tahitensis from different origins. Following the screening for odor-active molecules through gas chromatography-olfactometry and aroma extract dilution analysis (GC-O and AEDA), selected compounds were quantified using stable isotope dilution assays (SIDA) and their dose over threshold values (DoTs) were calculated. Vanillin was confirmed as the most important odor-active compound due to its highest DoT value, especially in the V. planifolia sample. Meanwhile, 4-methoxybenzyl alcohol and 4-methoxybenzaldehyde showed higher DoT factors than vanillin in V. pompona and partially in V. tahitensis samples. This indicates their role as discriminative odorants for these varieties. The heightened DoT values of 3-hydroxy-4,5-dimethyl-2(5H)-furanone in Uganda Vanilla samples unveil geographical influences on the odorant profile within V. planifolia species. Additionally, 2-methyl-3-(methyldithio)furan was identified for the first time in Vanilla samples with diverse DoT values from different species and origins.

A semi-mechanistic aging model of Pb added to soil by a modified stable isotope dilution technique

The aging of Pb added to soils has not been studied by the isotopic technology because of difficulties in determination of isotopically exchangeable Pb in soil, so that a set of 10 typical agricultural soils in China and a one-year aging experiment with the addition of water-soluble Pb to the soils were carried out. A modified stable isotope dilution technique to determine isotopically exchangeable Pb in soil was developed where 0.2 mM EDTA (ethylenediaminetetraacetic acid) as the extractant. When water-soluble Pb was added to soil, the isotopically exchangeable Pb (Eadd%, the percentage of isotopically exchangeable Pb to total Pb added to soil) initially decreased rapidly and gradually slowly. A semi-mechanistic aging model of Pb added to soils, including precipitation/nucleation (Y1), micropore diffusion (Y2), and organic matter encapsulation processes (Y3) was developed with the root mean square error 8.3% where Y1, Y2, and Y3 accounted for 0.02~26.9%, 1.4~21.8% and 3.8~11.3%, respectively, when the pH 4.0~8.0 and organic matter 2.0~6.0%. Soil pH was a vital factor affecting the aging rate. When the pH increased by 1 unit, the Eadd value decreased by approximately 9%. The model could be used to scale ecotoxicological data of Pb in soil generated in different aging times.

Garnet Reference Materials for In Situ Lu‐Hf Geochronology

In situ garnet Lu‐Hf geochronology has the potential to revolutionise the chronology of petrological and tectonic processes, yet there is a paucity of well‐characterised reference materials to account for laser‐induced matrix‐dependant elemental fractionation. Here, we characterise two reference garnets GWA‐1 (Lu ~ 7.0 μg g−1) and GWA‐2 (Lu ~ 8.5 μg g−1) for in situ garnet Lu‐Hf geochronology. Isochron ages from isotope dilution Lu‐Hf analyses yield crystallisation ages of 1267.0 ± 3.0 Ma with initial 176Hf/177Hfi of 0.281415 ± 0.000012 (GWA‐1), and 934.7 ± 1.4 Ma with 176Hf/177Hfi of 0.281386 ± 0.000013 (GWA‐2). In situ Lu‐Hf analyses yield inverse isochron ages up to 10% older than the known crystallisation age due to matrix effects between garnet and reference glass (NIST SRM 610) under different instrument tuning conditions. This apparent age offset is reproducible for both materials within the same session and can be readily corrected to obtain accurate ages. Our results demonstrate that GWA‐1 and GWA‐2 are robust reference materials that can be used to correct for matrix‐analytical effects and also to assess the accuracy of in situ Lu‐Hf garnet analyses across a range of commonly encountered garnet compositions.

Assessing a Fermented Whey Beverage Biofortified with Folate as a Potential Folate Source for Humans.

Folate, a vital water-soluble vitamin (B9), requires specific attention as its recommended daily intake frequently is not reached in countries without mandatory fortification. In this regard, biofortification with microorganisms like Bifidobacterium and Streptococcus offers a compelling approach for enhancing food with natural folates. A randomized, nonblinded, and monocentric human pilot study is conducted to assess the bioavailability of a folate-biofortified fermented whey beverage, comprising 3 intervention days and a controlled replenishment phase before and during the assay. Folate plasma concentration (5-CH3-H4folate) is determined using a stable isotope dilution assay and LC-MS/MS detection. Biokinetic parameters (cmax and tmax) are determined, and areas under the curve (AUC) normalized to the basal folate plasma concentration are calculated. An average bioavailability of 17.1% in relation to the 5-CH3-H4folate supplement, ranging from 0% to 39.8%, is obtained. These results reiterate the significance of additional research into folate bioavailability in general and dairy products. Further investigations are warranted into folate-binding proteins (FBP) and other potential limiting factors within the food and individual factors. In summary, biofortification via fermentation emerges as a promising avenue for enhancing the natural folate content in dairy and other food products.

Assessing Micronutrients in Soil with a New Chemical Extractant Using Isotopic Dilution Techniques: Linkages to Labile Fractions in Soil

ABSTRACT With the purpose of economizing the routine soil testing for micronutrients and avoiding the use of multiple soil extraction procedures, a laboratory investigation was attempted to evaluate a new chemical extractant for assessing the availability of common micronutrients (Fe, Cu, Mn, Zn, Mo, and B) in soils of widely varying properties (n = 32). The new extractant designated as OM-MES-VERA# contained 0.01 M oxalic acid, 0.02 M maleic acid, 0.02 M 2-(N-Morpholino) ethane sulfonic acid (MES), 0.0025 M ethylene diamine tetraacetic acid di-sodium salt, 0.02 M acetic acid, and 0.1% polyacrylamide (final pH adjusted to 6.0). OM-MES-VERA extracted significantly higher amounts of micronutrient cations and B from soils compared to the conventional diethylene triamine pentaacetic acid (DTPA, pH 7.3) extractable micronutrient cations and hot water soluble B, respectively, but lesser amount of Mo compared to Mehlich-3 universal extractant. Soil test values by both OM-MES-VERA- and conventional extractants were correlated with soil properties. The labile (E and L) values of micronutrient cations and Mo estimated by isotopic dilution method, ammonium bicarbonate-DTPA, pH 7.6 extractable soil B (as an estimate of E for B) and B concentration in maize (as an estimate of L for B) were successfully regressed on soil test values of micronutrients by OM-MES-VERA and conventional soil extractants except in the case of DTPA (pH 7.3) for E-value of Fe and L-value of Mn. The OM-MES-VERA extractant can be considered to assess the availability of common micronutrients in soils of diverse soil properties.

Combination of Standard Addition and Isotope Dilution Mass Spectrometry for the Accurate Determination of Melamine and Cyanuric Acid in Infant Formula.

In the melamine scandals of the early 2000s, different companies of the dairy industry cheated their products by applying chemical substances to feign a higher content of nitrogen. However, this had a severe toxic impact on the kidney health of consumers. As a result, tremendous effort was put into the prevention of further harm to the public. In the present study, a fast-screening method for the determination of melamine and cyanuric acid in infant formula was developed. While a 1D-LC approach is faster and easier to set up, a 2D-LC approach allows for a more accurate result with better selectivity and sensitivity. For both instrumental approaches, the signal ratio of the isotopologues was crucial and had a dominant effect on the results and the measurement uncertainty. For this reason, the different contributions to the measurement uncertainty were determined experimentally using Matched Standard Addition-IDMS and compared to the Exact Matching Double IDMS.

High-precision U-Pb zircon dating identifies a major magmatic event on the Moon at 4.338 Ga.

The Moon has had a complex history, with evidence of its primary crust formation obscured by later impacts. Existing U-Pb dates of >500 zircons from several locations on the lunar nearside reveal a pronounced age peak at 4.33 billion years (Ga), suggesting a major, potentially global magmatic event. However, the precision of existing geochronology is insufficient to determine whether this peak represents a brief event or a more protracted period of magmatism occurring over tens of millions of years. To improve the temporal resolution, we have analyzed Apollo 14, 15, and 17 zircons that were previously dated by ion microprobe at ~4.33 Ga using isotope dilution thermal ionization mass spectrometry. Concordant dates with sub-million-year uncertainty span ~4 million years from 4.338 to 4.334 Ga. Combined with Hf isotopic ratios and trace element concentrations, the data suggest zircon formation in a large impact melt sheet, possibly linked to the South Pole-Aitken basin.

Direct determination of the 237Np/238U ratio in nuclear fuel samples by multicollection inductively coupled plasma mass spectrometry (MC-ICP-MS)

A new method for the direct measurement of 237Np/238U ratio in irradiated UO2 pellets by multicollection inductively coupled plasma mass spectrometry (MC-ICP-MS) is proposed. It allows the determination of ratios down to 10 × 10–6 mol·mol−1 using ion counter and Faraday cup. This approach was validated by intercomparison with the usual two-step-method (Quadrupole ICP-MS for 237Np determination and isotope dilution mass spectrometry (IDMS) for 238U). For ratios between 10 × 10–6 and 100 × 10–6 mol·mol−1, expanded uncertainties (k = 2) varied from 2.75% to 0.81%, twice lower than the uncertainties determined by the usual method.

Characterization of typical transuranic nuclides in a reference fallout material using SF-ICP-MS

For the quality control in determining transuranic nuclides in fallout samples, this work first reported the 237Np activity concentration in a reference fallout material and further calculated the activity ratios of 237Np/239+240Pu and 237Np/241Am, and the atom ratio of 237Np/239Pu in it. The reference fallout material prepared by the Meteorological Research Institute was collected at 14 stations throughout Japan in 1963–1979. The 237Np and Pu isotopes (239Pu and 240Pu) were separated and purified using AG MP-1M anion-exchange resin, quantified using 242Pu as an isotope dilution tracer, and determined by the SF-ICP-MS. The analytical method was validated by the analysis of 4 sediment reference materials. The activity concentrations of 237Np, 239Pu and 240Pu were (25.9 ± 0.6) × 10−3, 4.10 ± 0.01 and 2.89 ± 0.04 Bq/kg, respectively, in the investigated reference fallout material. The activity ratio of 237Np/239+240Pu (3.7 ± 0.1) × 10−3 was consistent with the global fallout evaluation value. The 237Np/239Pu atom ratio of 0.561 ± 0.014 was higher than the average global fallout value of 0.41 ± 0.010, indicating the necessity of establishing regional characteristic global fallout value of 237Np/239Pu atom ratio for assessment of radioactive contamination. Comparison of the 237Np/239+240Pu activity ratios between in the reference fallout material and in soils over several decades indicated that 237Np has stronger migration capability than Pu isotopes in soils because 237Np was depleted compared to reference fallout material.

A candidate reference measurement procedure for quantification of glycocholic acid in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry

Accurate measurement of serum glycocholic acid (GCA) is crucial for evaluating the activity of chronic hepatitis. Moreover, GCA is a novel identified biomarker for hepatocellular carcinoma. Although some laboratories have used the liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to measure GCA in recent years, the problem of potential interference of GCA analogues has not been solved well yet. Neither reference measurement procedures nor reference materials for GCA have been listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. For standardization of GCA, it is urgent to establish a candidate measurement procedure for GCA. In this study, a candidate reference measurement procedure for the quantification of GCA in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC–MS/MS) by a two-step sample pretreatment of protein precipitation and MAX solid-phase extraction was developed and validated. GCA can be completely separated from its structural analogues with gradient elution in 9 min compared with short time gradients published in previous literature by Huang’s group. Method validation indicated perfect quantitation precision with intra-day and inter-day values that were ≤1.30% and ≤1.80%, respectively. The method showed excellent linearity with high regression coefficients (R2 > 0.999) over a range of 0.92 ng/g–38.38 μg/g and perfect recoveries at three spiked levels (99.87–100.43%). No interference, matrix effect, and carryover were observed. Moreover, the cRMP was successfully applied to measure GCA in serum samples and compared with two immunoassays in a clinical laboratory. As a candidate reference method, this method can promote a GCA standardization program.Graphical

Updating the Upper Cretaceous (Campanian) Two Medicine Formation of Montana: Lithostratigraphic revisions, new CA-ID-TIMS U-Pb ages, and a calibrated framework for dinosaur occurrences

The Campanian Two Medicine Formation of northwestern Montana, USA, is richly fossiliferous, and discoveries made within the unit over the past century have greatly advanced our appreciation of dinosaur paleobiology and evolution. Previously undifferentiated from a lithostratigraphic perspective, the formation is now subdivided into four new members that include (from base to top) (1) the Rock City Member, (2) the Shields Crossing Member, (3) the Hagans Crossing Member, and (4) the Flag Butte Member. These new formal units and their associated fossil occurrences are also now included in an age model founded on eight high-resolution chemical abrasion−isotope dilution−thermal ionization mass spectrometry (CA-ID-TIMS) U-Pb ages. New age data confirm that the Two Medicine Formation accumulated during much of the Campanian, with deposition spanning ca. 82.4 Ma to 74.4 Ma. New age data further indicate that a major reorganization of depositional systems, marked by a shift from predominantly lacustrine to alluvial facies and accompanied by a dramatic increase in accommodation, transpired near the base of the new Flag Butte Member at ca. 76.3 Ma. This change in depositional regime correlates in age with the Judith River−Belly River discontinuity, which marks the contact between the McClelland Ferry and Coal Ridge Members in the Judith River Formation and coincides with the onset of the Bearpaw transgression in north-central Montana. The new lithostratigraphic and chronostratigraphic framework for the Two Medicine Formation serves to contextualize and calibrate the formation’s rich dinosaur fossil record, which can now be interrogated with increased clarity and precision. These results also provide ground truth for numerical models that explore the structure of the fossil record in relation to alluvial architecture and terrestrial sequence stratigraphy.

Trends in acrylamide content in selected potato/sweet potato products on the Canadian market

Processed plant-based foods, particularly high carbohydrate-containing foods, are among the greatest contributors to dietary acrylamide, a probable human carcinogen, uptake. Between 2009 and 2020, five surveys were conducted to determine acrylamide in high carbohydrate-containing foods in Canada. These surveys included sampling of potato and sweet potato chips, French fries, and frozen potato/sweet potato products, as a follow-up to our earlier surveys from 2002 - 2008. Samples were analyzed using isotope dilution (13C3-acrylamide) with LC-MS/MS. The highest mean acrylamide levels were found in sweet potato chips. Among potato chips (57 to 4660 ng g−1), one brand consistently showed the highest concentrations with wide variability. Acrylamide concentrations decreased over time in ready-to-eat French fries (from 480 to 358 ng g−1), and one brand showed a clear reduction temporally. Wide variations were observed among brands, among lots/outlets of same brands, and among different food chains. Acrylamide levels in potato chips decreased between 2009 and 2016 (504.3 ng g−1) relative to the period 2002 - 2008 (1096.9 ng g−1). The acrylamide trends observed in the products measured in the latest study indicate that food producers may have adopted mitigation strategies.

Measurement of iron absorption and iron gains from birth to 6 months in breastfed and formula-fed infants using iron isotope dilution.

Little is known about iron kinetics in early infancy. We administered stable iron isotopes to pregnant women and used maternal-fetal iron transfer to enrich newborn body iron. Dilution of enriched body iron by dietary iron with natural isotopic composition was used to assess iron kinetics from birth to 6 months. In breastfed (BF, n = 8), formula-fed (FF, n = 7), or mixed feeding (MF, n = 8) infants, median (interquartile range) iron intake was 0.27, 11.19 (10.46-15.55), and 4.13 (2.33-6.95) mg/day; iron absorbed was 0.128 (0.095-0.180), 0.457 (0.374-0.617), and 0.391 (0.283-0.473) mg/day (BF versus FF, P < 0.01); and total iron gains were 0.027 (-0.002-0.055), 0.349 (0.260-0.498), and 0.276 (0.175-0.368) mg/day (BF versus FF, P < 0.001; BF versus MF, P < 0.05). Isotope dilution can quantify long-term iron absorption and describe the trajectory of iron depletion during early infancy.

Comparative study for purity assessment of recombinant human growth hormone using mass balance approach and amino acid-based isotope dilution mass spectrometry

Growth hormone plays an unreplaceable role in regulating human growth and promoting protein synthesis, etc. For this reason, accurate quantification of its purity has become a focus of attention in the pharmaceutical industry and medical research. Herein, the purity assessment of human growth hormone (hGH) was established on the basis of mass balance approach and amino acid (AA)-based isotope dilution mass spectrometry (IDMS). In the first method, hGH purity was quantified by the determination of all the impurities in a hGH study material. In the second method, hGH purity was assessed by quantifying hydrolyzed AAs, e.g. leucine (Leu), phenylalanine (Phe), and proline (Pro). To accurately quantify these AAs, the corresponding isotope-labeled AAs were used as internal standards using high-performance liquid chromatography- mass spectrometry (HPLC-MS). The results of the two methods for determining the purity of hGH were consistent, with the mass balance method giving a result of (0.352 ± 0.010) g/g and IDMS a result of (0.363 ± 0.028) g/g. The work in this study could give a benefit for protein quantification and provides a reference for the development of hGH certified reference materials.

Discrimination of overheated pasteurized milk using mass spectrometry-based proteomics

Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides—INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK—were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

Determination of capsaicin at trace levels in different food, biological and environmental samples by quadruple isotope dilution-gas chromatography mass spectrometry after its preconcentration

Despite the therapeutic properties of capsaicin for some diseases, it shows some side effects for human health. The goal of this study was to develop a precise and accurate analytical strategy for the trace determination of capsaicin in different food, biological and environmental samples including pepper, saliva and wastewater by gas chromatography–mass spectrometry (GC–MS) after spraying-based fine droplet formation-liquid phase microextraction (SFDF-LPME) and quadruple isotope dilution (ID4) method. Acetic anhydride was used as derivatizing agent, and the extraction method was used to enrich the analyte derivative to reach low detection limits. Under the optimum conditions, limit of detection (LOD) and limit of quantitation (LOQ) were determined to be 0.33 and 1.10 µg/kg, respectively. Percent recoveries calculated for SFDF-LPME-GC-MS method ranged between 84.1 and 131.7 %. After the application of ID4-SFDF-LPME-GC-MS method, percent recoveries were obtained in the range of 94.9 and 104.0 % (%RSD ≤ 2.8) for the selected samples. It is obvious that the isotope dilution-based method provided high accurate and precise results due to the elimination of errors during the derivatization, extraction and measurement steps.

Is the TCA cycle malate dehydrogenase-citrate synthase metabolon an illusion?

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques.