Sally A. Schuette

Feasibility of using the stable isotope 25Mg to study Mg metabolism in infants.

ABSTRACT: The feasibility of using isotopic techniques to study Mg absorption and metabolism was explored in three full-term human infants. 25Mg (98.8 atom %) was administered orally as an in vivo tracer. Fractional 25Mg absorption, isotope retention, endogenous fecal Mg losses, and apparent Mg exchangeable pool size were then determined under three conditions of isotope administration: 1) 20 mg 25Mg, with single feeding; 2) 20 mg 25Mg, distributed over a 24-h period; and 3) 60 mg 25Mg, over a 24-h period. Mg isotope ratios were determined by inductively coupled plasma mass spectrometry. Fractional absorption was increased in all three infants after distributed versus bolus administration at the 20 mg dose; mean (±SD) fractional absorption was 64.0 ± 3.9 versus 54.3 ± 5.9%, respectively. 25Mg retention was also more in all three infants after distributed administration (55.8 ± 3.0 versus 44.3 ± 1.3% of dose). At the 60-mg 25Mg dose, compared to 20 mg, fractional absorption was reduced but absolute isotope absorption more than doubled in all infants; urine isotope losses represented a similar fraction of the absorbed dose, thus, 25Mg retention also more than doubled. Compared to the results of the isotope studies, net Mg absorption and balance were uninfluenced by total Mg intake. Isotope retention with distributed isotope administration resulted in measurable isotopic enrichment of plasma and erythrocytes at 72 h (i.e. plasma isotope enrichment was 6.3-10.2 and 19.2-23.5% for the 20- and 60-mg dose, respectively). With these doses, apparent Mg exchangeable pool size ranged from 5.5 to 7.6 mmol/kg body wt; these values showed a decrease with age both within and between infants. Our results indicate that Mg stable isotope studies may offer sufficient accuracy and reproducibility to permit meaningful investigations of Mg bioavailability and developmental changes in Mg metabolism in human infants.

Accurate measurement of stable isotopes of magnesium in biological materials with inductively coupled plasma mass spectrometry

A method is described for the accurate isotopic determination of magnesium (24Mg, 25Mg, 26Mg) in biological materials, which is based on inductively coupled plasma mass spectrometry (ICP-MS). The analytical performance of the method was examined with respect to the requirements of stable isotope tracer studies. When applied to the measurement of base-line isotope ratios (MRa/b) in rat tissue the following results were obtained (data are given in terms of MR25/24 and MR26/24± 1RSD, n= 4 or 5): standard solution of Mg (0.05 µg ml–1), 0.1394 ± 0.6, 0.1697 ± 0.8; bone, 0.1401 ± 0.5, 0.1706 ± 0.8; brain, 0.1404 ± 0.2, 0.1720 ± 0.3; kidney, 0.1392 ± 0.5, 0.1702 ± 0.6; liver, 0.1388 ± 0.4, 0.1696 ± 0.5; muscle, 0.1396 ± 0.2, 0.1716 ± 0.4; plasma, 0.1385 ± 0.3, 0.1691 ± 0.3; red cells, 0.1383 ± 0.1, 0.1694 ± 0.2; and urine, 0.1402 ± 0.1, 0.1721 ± 0.3. The measurement precision for replicate analyses of each matrix was in the range 0.1–1.0% and the mean value of the isotope ratio for different matrices agreed with the corresponding ratio for the standard solution to within 1.5%. When measured over a 10-h period, the isotope ratios appeared to be independent of the observed drifts in the ion beam intensities. The instrument blank contribution to the ion beam intensities was about 0.1% of the values obtained for a solution containing 0.05 µg ml–1 of natural Mg. The absolute detection limit for Mg (based on the experimental standard deviation of blanks run over 10 h) was <2 ng for all three stable isotopes. The ICP-MS phase of the analysis was capable of making 50–150 separate measurements of both ratios in a 10-h period, depending on the desired level of measurement precision within the range 0.1–1.0%. A complete analytical scheme for the accurate determination of the three stable isotopes of Mg, which is based on precipitation with ammonium phosphate, is described. The accuracy of the method was tested using the standard reference materials Bovine Liver (NBS 1577a, 600 ± 15 µg g–1 of Mg) and Animal Bone (IAEA H-5, 3550 ± 90 µg g–1). The proposed method provided the following data (µg g–1): SRM 1577a, 617 ± 4 and IAEA H-5, 3585 ± 16. For other biological matrices of interest, the accuracy of the method was compared with atomic absorption spectrometry. The complete analytical procedure, up to the point of mass spectrometric measurement, can be performed on about 20 samples per working day.

Bioavailability of Magnesium Diglycinate vs Magnesium Oxide in Patients with Ileal Resection

BACKGROUND Patients who have undergone ileal resection are at risk for developing magnesium depletion/deficiency because of poor absorption and decreased intake as well as increased endogenous losses. Magnesium repletion is difficult to accomplish because of the cathartic action of most oral magnesium supplements at therapeutic doses. The results of in vitro and in situ studies show that magnesium diglycinate (chelate) represents a highly available form of magnesium that is absorbed in part as an intact dipeptide in the proximal small intestine. METHODS We conducted a double-blind, randomized crossover trial with 12 patients who had ileal resections in order to compare the bioavailability of a 100-mg dose of 26Mg-labeled chelate with MgO in this patient population. RESULTS For the patient group as a whole, 26Mg absorption was low but was not different for the two supplements (23.5% vs 22.8% for magnesium chelate and MgO, respectively). However, 26Mg absorption was substantially greater from the chelate (23.5% vs 11.8%; p < .05) in the four patients who showed the greatest impairment of magnesium absorption with MgO and was better tolerated by all patients. Peak isotope enrichment also occurred significantly earlier after 26Mg chelate than after 26MgO ingestion (mean difference 3.2 +/- 1.3 hours; p < .05), and the area under the enrichment vs time curve was greater after chelate ingestion (p < .05). CONCLUSIONS Data from this study support the suggestion that some portion of magnesium diglycinate is absorbed intact, probably via a dipeptide transport pathway. Magnesium diglycinate may be a good alternative to commonly used magnesium supplements in patients with intestinal resection.

Dysprosium chloride as a nonabsorbable gastrointestinal marker for studies of stable isotope-labeled triglyceride excretion in man.

Objective: The aim of this work was to determine if dysprosium chloride (DyCl3) is a suitable nonabsorbable marker for studies of labeled-triglyceride excretion in cystic fibrosis patients allowing excretion to be determined accurately after analysis of one or two stools. Methods: A series of 66 absorption studies were conducted in 36 young cystic fibrosis patients over a five year period. All tests consisted of ingesting a single test meal containing both 13C-labeled triglyceride (TG*) and DyCl3; in most studies the food colorant brilliant blue (FD&C blue #1) was administered along with the DyCl3. Ingestion of the test meal was followed by collection of individual stools for 72 to 96 hours. Stools were analyzed for 13C-Excess (13C*) and Dy. Results: Excretion of Dy in cystic fibrosis patients who exhibited a wide-range of steatorrhea was quantitative. Fractional excretion of Dy and 13C* in individual stools showed a high linear correlation (r2 = 0.969) with a slope and y-intercept close to unity and zero, respectively. As a result, estimates of TG* excretion based on analysis of only two stools (partial pool method, PPM) were not different from those based on the analysis of all stools or stool composites. This was true both when Dy content and when stool color due to ingested brilliant blue was used to determine which stools to analyze for the PPM. Conclusions: Combining the use of Dy and brilliant blue permits reasonably accurate estimates of fecal TG* excretion after analysis of samples from two easily identified stools. This practical method can be used to address many important clinical and experimental questions regarding triglyceride digestion and absorption that may otherwise go unanswered.

[13C]Glucose Breath Testing Provides a Noninvasive Measure of Insulin Resistance: Calibration Analyses Against Clamp Studies

BACKGROUND Exhaled (13)CO2 following ingestion of [(13)C]glucose with a standard oral glucose tolerance load correlates with blood glucose values but is determined by tissue glucose uptake. Therefore exhaled (13)CO2 may also be a surrogate measure of the whole-body glucose disposal rate (GDR) measured by the gold standard hyperinsulinemic euglycemic clamp. SUBJECTS AND METHODS Subjects from across the glycemia range were studied on 2 consecutive days under fasting conditions. On Day 1, a 75-g oral glucose load spiked with [(13)C]glucose was administered. On Day 2, a hyperinsulinemic euglycemic clamp was performed. Correlations between breath parameters and clamp-derived GDR were evaluated, and calibration analyses were performed to evaluate the precision of breath parameter predictions of clamp measures. RESULTS Correlations of breath parameters with GDR and GDR per kilogram of fat-free mass (GDRffm) ranged from 0.54 to 0.61 and 0.54 to 0.66, respectively (all P<0.001). In calibration analyses the root mean square error for breath parameters predicting GDR and GDRffm ranged from 2.32 to 2.46 and from 3.23 to 3.51, respectively. Cross-validation prediction error (CVPE) estimates were 2.35-2.51 (GDR) and 3.29-3.57 (GDRffm). Prediction precision of breath enrichment at 180 min predicting GDR (CVPE=2.35) was superior to that for inverse insulin (2.68) and the Matsuda Index (2.51) but inferior to that for the log of homeostasis model assessment (2.21) and Quantitative Insulin Sensitivity Check Index (2.29) (all P<10(-5)). Similar patterns were seen for predictions of GDRffm. CONCLUSIONS (13)CO2 appearance in exhaled breath following a standard oral glucose load with added [(13)C]glucose provides a valid surrogate index of clamp-derived measures of whole-body insulin resistance, with good accuracy and precision. This noninvasive breath test-based approach can provide a useful measure of whole-body insulin resistance in physiologic and epidemiologic studies.

Intra-individual variability of CO2 breath isotope enrichment compared to blood glucose in the oral glucose tolerance test

BACKGROUND Glucose tolerance can be assessed noninvasively using (13)C-labeled glucose added to a standard oral glucose load, by measuring isotope-enriched CO(2) in exhaled air. In addition to the clear advantage of the noninvasive measurements, this approach may be of value in overcoming the high variability in blood glucose determination. METHODS We compared within-individual variability of breath CO(2) isotope enrichment with that for blood glucose in a 75-g oral glucose tolerance test (OGTT) by adding 150 mg of d-[(13)C]glucose ((13)C 99%) to a standard 75-g glucose load. Measurements of whole blood glucose (by glucose oxidase) and breath isotope enrichment (by isotope ratio mass spectrometry) were made every 30  min for 3 h. Subjects underwent three repeat tests over a 3-week period. Values for variability of breath isotope enrichment at 3 h (∂‰180) and of area under the curve for enrichment to 180  min (AUC180) were compared with variability of the 2-h OGTT blood glucose. RESULTS Breath test-derived measures exhibited lower within-subject variability than the 2-h OGTT glucose. The coefficient of variation for ∂‰180 was 7.4 ± 3.9% (mean ± SD), for AUC180 was 9.4 ± 6.3%, and for 2-h OGTT blood glucose was 13 ± 7.1% (P = 0.005 comparing ∂‰180 versus 2-h blood glucose; P = 0.061 comparing AUC180 versus 2-h blood glucose; P = 0.03 comparing ∂‰180 versus AUC180). CONCLUSIONS Breath test-derived measurements of glucose handling had lower within-subject variability versus the standard 2-h blood glucose reading used in clinical practice. These findings support further development of this noninvasive method for evaluating glucose tolerance.

Feasibility of measuring organ magnesium turnover in vivo by continuous feeding of a stable isotope

Abstract The feasibility of measuring organ endogenous magnesium (Mg en ) turnover in vivo by continuous feeding of a single stable isotope of Mg was demonstrated in this investigation. Adult CD-1 mice were fed a Mg deficient diet and deionized water with (+Mg) or without (−Mg) added 24 Mg (290 μg/mL) for 16 days. The change in organ 25 Mg content over time was then accurately determined by in vitro isotope dilution with 26 Mg as spike. Organ endogenous Mg content was then calculated as 25 Mg en / 0.1028 and exogenous Mg (Mg ex ) content from the expression Mg ex = Mg total - Mg en . All soft tissues examined in the +Mg group showed significant turnover of Mg en and accumulation of Mg ex . The rate at which this occurred was organ specific. Apparent half-lives for Mg en turnover were 3.83, 4.13, 5.87, and 8.77 days for liver, heart, brain, and skeletal muscle, respectively. Mg restriction resulted in a dramatic decrease in the rate of Mg en turnover with apparent half-lives ranging from 60.3 to 146 days. Brain showed the smallest decrease in Mg en turnover with Mg restriction, and was the only tissue observed to lose a significant amount of total Mg.

Effect of Mg nutriture on the dynamics of administered 25Mg exchange in vivo in the rat.

The effect of Mg nutriture on Mg exchange and interorgan distribution was studied in adult rats ten days after a single I.P. dose of (25)Mg ( approximately 5 mg). First the effects of level of Mg intake (0.25, 0.05, or 0.01% Mg) on standard measures of Mg nutriture were studied for 62d to fully document the Mg status of the adult rats. The Mg-deficient diet led to a reduction in plasma, erythrocyte and urine Mg concentration but the only tissues affected were kidney and bone; no outward signs of deficiency were observed. At this point, the 4 remaining rats from each diet group received a single dose of (25)Mg and were killed 10d later. Unlike measures of total Mg content, Mg restriction was observed to significantly alter the distribution of isotope within the soft tissue compartment. The proportion of retained isotope accumulated by soft tissues other than skeletal muscle increased. Because this was not true for skeletal muscle, exogenous (25)Mg label was diverted to more metabolically active tissues during Mg restriction. The apparent Mg exchangeable pool (MgEP) size, determined by in vivo stable isotope dilution, reflected this difference in skeletal muscle (25)Mg accumulation; MgEP size was 39% lower in Mg restricted (0.01% Mg) compared to control (0.05% Mg) rats. The pool of exchangeable Mg in bone was also reduced by Mg restriction but, unlike the soft tissue compartment, the reduction in bone exchangeable Mg was quantitatively similar to the reduction in total Mg content.

The effect of diets high in fat and/or fiber on colonic absorption of DMH in the rat

The possibility that long-term feeding of diets high in fat or fiber could alter the colonic mucosa and subsequent colonic absorption of 1,2-dimethylhydrazine (DMH) in situ was examined in the rat model. Male Sprague-Dawley rats were fed one of four experimental diets for six weeks prior to studies of DMH absorption and bile acid excretion; dietary treatments consisted of two levels of fat (12 and 47% of calories from corn oil) fed at each of two levels of fiber (plus or minus 15% wheat bran). Two sets of DMH absorption studies (Studies 1 and 2) were performed; the first used a 10- and the second a 20-minute test period. In Study 1, DMH absorption was greater in those animals that had been fed the high level of corn oil when additional fiber was not present in the diet. When a longer absorption period was used (Study 2), this effect of diet on DMH absorption was not apparent. The level of fiber, not the fat intake, altered bile acid excretion. Bile acid concentration (mg/g dry wt) decreased with added fiber, whereas total bile acid excretion (mg/day) increased. These results indicate that high levels of dietary fat may result in small increases in DMH absorption which are unrelated to changes in bile acid concentration.

Effect of Triglyceride Structure on Fecal Excretion of 13C-Labeled Triglycerides

Objective: The aim of this work was to determine the effects of specific changes in the structure of 13C-labeled triglyceride (TG*) on its fecal excretion relative to total stool fat excretion determined simultaneously in patients with reduced exocrine pancreatic function. Methods: A series of 47 studies were conducted in 26 young cystic fibrosis (CF) patients and 11 adult patients with chronic pancreatitis over a five year period. Each test consisted of ingesting a single high fat test meal containing both 13C-labeled triglyceride (TG*) and dysprosium chloride (DyCl3) a nonabsorbable marker of intestinal transit; in most studies the food colorant brilliant blue (FD&C blue #1) was administered along with the DyCl3. The TG*s tested were: P*P*P* = TRIPALMITIN-1,1,1-13C3; SO*S = 2-OCTANOYL-1,3-DISTEARIN-2-octanoyl-1,2-13C2; and P*LP* = 2-LAURYL-1,3-DIPALMITIN-dipalmitoyl-1,1,2,2-13C4. Ingestion of the test meal was followed by collection of individual stools for at least 72 hours. Stools were analyzed for 13C-Excess (13C*), total fat, and Dy. Results: Excretion of P*LP* showed a high degree of linear correlation with stool fat (r2 = 0.924) over a wide-range of fecal fat values. Excretion of SO*S was also significantly correlated with stool fat, but its excretion was less than 10% at all levels of steatorrhea and the slope of the regression line relating TG* excretion to stool fat was some four to five times smaller than observed for P*LP*. Fecal excretion of P*P*P* was highly correlated with stool fat (r2 = 0.941) in patients with moderate steatorrhea (<25 g fat/24 hours) and the slope of the regression line (3.20) was considerably greater than for P*LP*. Only results from those studies in which stool collections were complete (Dy excretion >90%) were utilized in the statistical comparisons (36 of 47 studies). Conclusions: The observed highly significant linear correlation between P*LP* and stool fat over the entire range of steatorrhea suggests that P*LP* excretion may be a suitable surrogate for fecal fat in patients with reduced exocrine pancreatic function. Because fecal excretion of TG* administered as described can be accurately determined by sampling only two visually marked stools, development of a noninvasive test to replace the current 72-hour stool fat test using this approach is possible. Use of other engineered TG*s and/or labeled fatty acids, may provide a method for non-invasive in vivo assessment of the specific defect(s) leading to steatorrhea in other patient groups.