Single Isotope Method Research Articles

Exploring the differences of moisture traceability methods based on MixSIAR model under different nitrogen applications of wheat in the Arid Region of Northwest China

The Bayesian stable isotope mixing (MixSIAR) model was widely used in water source tracing. However, double isotope and single isotope calculated by the MixSIAR model led to different results in terms of previous studies. The effect of different nitrogen treatments on the water traceability results of wheat crops is still unclear. This study investigated the wheat root water uptake patterns at different nitrogen application gradients, as well as at different isotopes in the MixSIAR model. The results showed the main soil water uptake layer was relative constant at the treatment of 15% nitrogen reduction, and the water source was mainly from 0–10 cm soil layer from jointing to harvest. The main soil water uptake layer significantly varied at the treatment of 30% nitrogen reduction, which yielded the highest. 45% reduction of nitrogen treatment showed the deepest soil water uptake absorption on average with the whole wheat growth period. The consistency of the results between the two single isotope methods was better in the early stage of wheat and worse in the later stage. The inconsistency of root water uptake distribution results between the two single isotope methods might be influenced by the water transport mechanism of wheat stem and sampling errors. The double isotope method had the lowest uncertainty, but it might amplify the error of the sampling process. The uncertainty of the single hydrogen isotope method was less than single oxygen isotope method. This study provided a new evaluation of hydrogen-oxygen stable isotope traceability methods for wheat under different nitrogen treatments, and gave more ideas and insights for subsequent crop water traceability by the MixSIAR method.

Reference range for 24-h urine calcium, calcium/creatinine ratio, and correlations with calcium absorption and serum vitamin D metabolites in normal women.

Recommendations for a normal range for 24-h urine calcium vary from a low of 50 mg to a high of 400 mg; most "laboratory normal ranges" based on older literature are incorrect. The objective of this analysis is to define a normal range for young women age 25-45 years and older women age 55-90 years, white and black, for 24-h urine calcium and calcium/creatinine ratio, and to examine the relationship between 24-h urine calcium, calcium absorption, and vitamin D metabolites. Data from 3 studies was collected on 959 normal black and white women, ages 25-87 years, for 24-h urine calcium, creatinine, calcium intake, serum 25-hydroxyvitamin D (25OHD),1,25 dihydroxyvitamin D (1,25(OH)2D), and calcium absorption. Urine calcium and creatinine were measured on an auto-analyzer. Calcium absorption was measured by a single isotope method. Serum 25OHD and 1,25(OH)2D were measured by competitive protein binding assay or radioimmunoassay. Because 24-h urine calcium did not fit a normal distribution, non-parametric methods were used to determine the 95% reference interval (RI) and 90% non-parametric confidence intervals (CI) calculated for groups. The 95% reference intervals for 24-h urine calcium for women were as follows: black older 7-225 mg, black younger 8-285 mg; white older 37-275 mg, and white younger 23-287 mg. In older white women, 24-h urine calcium was significantly correlated with serum 1,25(OH)2 D, r = 0.23, p < 0.001, calcium intake r = 0.12, p = 0.001, and calcium absorption r = 0.18, p = 0.003, but not serum 25OHD r = 0.07, p = 0.06. The normal reference interval for 24-h urine calcium for black women is lower than white women. Twenty-four-hour urine calcium was correlated with serum 1,25(OH)2 D calcium intake and calcium absorption, but not serum 25OHD. This range will be useful clinically for defining hypercalciuria and for following patients on vitamin D and calcium treatment.

Vegetation and location of water inflow affect evaporation in a subtropical wetland as indicated by the deuterium excess method

AbstractEvaporation represents a principal form of water loss from water bodies, but quantifying evaporation with transpiring vegetation is challenging, owing to the difficulties in disentangling evaporation from transpiration. As the two processes have different effects on water isotope signatures (i.e., oxygen and hydrogen stable isotope ratios expressed by δ18O and δ2H), we used both the δ18O and δ2H values of water samples to calculate their deuterium excess (d) values, which are proportionate to fraction of evaporation loss (Ef) of water being sampled. Comparing with a single isotope method, the d method holds the advantage that it does not require knowledge of the initial isotope ratios of source water. Limitations of the d method, for example, effects of prior‐evaporation of water before entering water bodies, can be checked and corrected. The sampling locations throughout our study site differed in vegetation coverage, water depth, and distance to water inflow (or discharge gate of water inflow), which are three important factors in water management. This sampling design offered us a unique opportunity to quantify the influences of the above three factors on Ef. We found the following: (1) There was a spatiotemporal pattern in δ18O distribution. (2) The Ef was negatively related to the vegetation coverage, positively related to distance to the water source inflow but nonsignificantly related to water depth. Therefore, vegetation and distance to the water inflow are two important considerations for the control of evaporative water loss, and evaporation calculated by the d method can lead to more informed water management.

Vitamin D Does Not Increase Calcium Absorption in Young Women: A Randomized Clinical Trial

It is commonly said that vitamin D should be used to increase calcium absorption. We tested this statement in a dose-response study of vitamin D on calcium absorption. A total of 198 white and African American women, aged 25 to 45 years, with vitamin D insufficiency, serum 25-hydroxyvitamin D (25OHD) <20 ng/mL, were randomized in a double-blind study to vitamin D3 400, 800, 1600, 2400 IU, or placebo. A calcium supplement was given to increase mean calcium intake at baseline from 706 mg/d to 1031 mg/d. Calcium absorption was measured at baseline and after 12 months using a single isotope method with radiocalcium45 and 100 mg of calcium. Mean baseline serum 25OHD was 13.4 ng/mL (33.5 nmol/L) and increased to 40 ng/mL (100 nmol/L) on the highest dose of 2400 IU. Using a multivariate regression analysis with significant predictors, baseline absorption, calcium intake, and weight, there was no increase in 12-month calcium absorption compared with baseline on any dose of vitamin D in either whites or African Americans. There was no significant relationship between 12-month calcium absorption and final serum 25OHD. In an analysis of calcium absorption and serum 25OHD at baseline, serum 25OHD levels were divided into groups: 0 to 5, 6 to 10, 11 to 15, or 16 to 20 ng/mL. There was no evidence of a threshold decrease in calcium absorption or serum 1,25 dihydroxyvitamin D (1,25(OH)2 D) amongst the lowest groups. Vitamin D doses up to 2400 IU daily did not increase calcium absorption. No threshold level of serum 25OHD for calcium absorption was found at baseline or in the longitudinal study, suggesting that active transport of calcium is saturated at very low serum 25OHD levels <5 ng/mL. There is no need to recommend vitamin D for increasing calcium absorption in normal subjects. Very efficient calcium absorption at very low levels of serum 25OHD explains why people do not develop osteomalacia provided that dietary intakes of calcium and phosphorus are adequate.

Blood Volume Determination, A Nuclear Medicine Test in Evolution

The method for determining blood volume has evolved substantially since first attempts were made in the latter part of the nineteenth century with the exsanguination of animals. The now accepted methods are based on indicator dilution methodologies. First attempts utilized inert dyes such as Evans Blue and Cardiogreen. These were found to be impractical due, primarily, to their rapid clearance from the blood. For many years, the most accepted method for blood volume determination was the dual isotope technique. This procedure utilizes chromium 51 or 99mTc to label autologous red cells and radioiodine 125 or 131 to label human serum albumin (HSA). Plasma and red cell volumes are measured separately and the results "combined". The procedure requires on-site labeling of autologous red cells and HSA, and meticulous preparation of standards and doses. The complexity of this method leads to performance times of 6 to 8 hours. An FDA-approved single isotope method is now employed in over 60 major institutions. HSA is labeled with radioiodine 131 at an FDA radiopharmaceutical facility, and test doses and standards are provided to laboratories in kit form. The red cell volume is derived by a calculation utilizing the measured plasma volume and the value for the average whole-body hematocrit. All calculations are carried out by a dedicated microprocessor, and a final report is generated and printed. The results are compared with predicted normal values for male and female patients based on percentage deviation from normal weight. Preliminary results are available in 30 minutes and complete calculations in 90 minutes.

Serum vitamin D metabolites and intestinal calcium absorption efficiency in women

Vitamin D sufficiency may be determined by the serum concentration of 25-hydroxyvitamin D [25(OH)D] that results in maximal intestinal calcium absorption efficiency. However, some investigators questioned whether 25(OH)D concentrations above the concentrations associated with rickets or osteomalacia influence calcium absorption. We determined whether calcium absorption efficiency is related to serum 25(OH)D or serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] concentrations. We measured calcium absorption efficiency in 492 black and white healthy women (age range: 20-80 y) by the single-isotope method with (45)Ca. Serum 25(OH)D concentrations were measured by a radioreceptor assay. Other relevant measurements included concentrations of serum 1,25(OH)(2)D, serum parathyroid hormone, serum creatinine, and serum estradiol, calcium intake, and bone mineral density. There was no relation between serum 25(OH)D concentrations and calcium absorption efficiency. In development of a multivariate model, the 4 major determinants of calcium absorption efficiency were menopausal status, calcium intake, and serum estradiol and serum 1,25(OH)(2)D concentrations. There was an interaction between serum 25(OH)D and 1,25(OH)(2)D concentrations on calcium absorption efficiency. The relation between calcium absorption and 1,25(OH)(2)D was positive, and this relation was stronger for lower concentrations of 25(OH)D than for higher concentrations of 25(OH)D. The relation of serum 25(OH)D to calcium absorption is not useful as an indicator of vitamin D sufficiency.

Validation of a single‐isotope‐labeled cholesterol tracer approach for measuring human cholesterol absorption

Cholesterol absorption is frequently determined using the plasma dual stable-isotope ratio method (PDSIRM). However, this method involves intravenous injection of stable-isotope-labeled cholesterol with simultaneous oral administration of differently labeled cholesterol, which results in high study costs and involves additional ethical considerations. The objective of the present study was to validate a simpler single-isotope method for determining cholesterol absorption against PDSIRM by using data from two previous studies. Enrichments of carbon-13 (13C and deuterium in red blood cells were analyzed by using differential isotope ratio MS. The area under the curve of 13C-enrichment in the plasma free-cholesterol pool was found to be significantly correlated with cholesterol absorption measured by using PDSIRM for study 1 (r = 0.85, P < 0.0001) and study 2 (r = 0.81, P < 0.0001). Average 13C-enrichment correlated with the area under the curve of 13C-enrichment in the plasma free cholesterol for both study 1 (r = 0.98, P < 0.0001) and study 2 (r = 1.00, P < 0.0001). Study 1 examined the efficacy and mechanisms of unesterified plant sterols and stanols on lipid profiles in hypercholesterolemic men and women, while study 2 investigated the effects of phytosterol vs. phytostanol esters on plasma lipid levels and cholesterol kinetics in hyperlipidemic men. Experimental approaches to determine cholesterol absorption were identical between the two studies. Consequently, in both studies, correlations (r = 0.88, P < 0.0001 for study 1, and r = 0.82, P < 0.0001 for study 2) were found between the average 13C-enrichment of plasma free cholesterol and cholesterol absorption measured by PDSIRM. These results suggest that a single-isotope-labeled cholesterol tracer approach can be used as a reliable noninvasive method to replace PDSIRM for examining changes in cholesterol absorption.

Ursodeoxycholic acid enhances fractional calcium absorption in primary biliary cirrhosis.

Bone disease is a frequently reported complication in primary biliary cirrhosis (PBC), but its pathogenesis is poorly understood. Calcium malabsorption has been considered as an important contributing factor. Ursodeoxycholic acid (UDCA) is the treatment of choice in PBC, improving survival, but its effect on calcium absorption is unknown. In this study, we have measured fractional calcium absorption, using a single isotope method, in a group of female PBC patients (median age: 60 years, range: 46-78 years) and age-matched female controls (median age: 58 years, range: 36-74). Bone mineral density (BMD) in PBC patients was significantly lower than age-matched controls (g/cm(2) +/- SEM; lumbar spine: controls 1.139+/-0.028, PBC patients 1.004+/-0.026, p = 0.0028; femoral neck: controls 0.944+/-0.034, PBC patients 0.819+/-0.023, p = 0.0032). Twenty two PBC patients, who were not vitamin D-deficient, were off and on UDCA for approximately 1 month and approximately 8 weeks, respectively. Fractional calcium absorption in PBC patients prior to UDCA treatment (mean +/- SEM, 33.8+/-2.6%) was significantly lower than controls (52.0+/-2.4%, p<0.001). Following UDCA therapy, fractional calcium absorption increased significantly (Off UDCA: 33.1+/-2.6%, On UDCA: 36.6+/-2.5%, p<0.0058). Osteocalcin levels were significantly raised in the PBC group (mean +/- SEM, ng/ml, 41.4+/-2.02) compared to controls (31.1+/-2.64, p = 0.002). There were no differences in parathyroid hormone (PTH) or 25-hydroxyvitamin D levels between these two groups or following UDCA therapy. In conclusion, we found that PBC patients display low spinal and femoral neck BMD, reduced fractional calcium absorption, and elevated plasma osteocalcin. The calcium malabsorption is corrected partially by UDCA therapy. Long-term studies are required to determine whether this effect can be sustained, and whether a sustained increase in fractional calcium absorption can translate into a favorable change in bone strength in patients with PBC.

Smoking Increases Bone Loss and Decreases Intestinal Calcium Absorption

Cigarette use is a risk factor for increased bone mineral density (BMD) loss but the mechanisms are not well understood. The relationship of smoking to rates of BMD change at the femoral neck, spine, and total body, and to intestinal calcium absorption were examined in 402 elderly men and women (32 smokers, 370 nonsmokers) who participated in a 3-year placebo-controlled study of calcium and vitamin D supplementation. Subjects in the supplemented group took 500 mg/day of elemental calcium and 700 IU/day of cholecalciferol. Two-hour calcium absorption fraction was determined three times, at 18, 30, and 36 months, with a single isotope method utilizing45Ca in a subset of 333 subjects. Annualized rates of BMD loss (adjusted for baseline BMD, weight, age, gender, supplementation status, and dietary calcium intake) were higher in smokers than nonsmokers at the femoral neck (−0.714 ± 0.285 %/year vs. +0.038 ± 0.084 %/year, p < 0.02), and total body (−0.360 ± 0.101 %/year vs. −0.152 ± 0.030 %/year, p < 0.05). No significant difference was observed at the spine (+0.260 ± 0.252 %/year in smokers vs. +0.593 ± 0.074 %/year in nonsmokers, p = 0.21). The mean (± SEM) calcium absorption fraction was lower in smokers (12.9 ± 0.8%, n = 23) than nonsmokers (14.6 ± 0.2%, n = 310, p < 0.05) after adjustment for gender, age, supplementation status, and dietary calcium and vitamin D intakes. Smokers of at least 20 cigarettes per day (n = 15) had the lowest mean absorption fraction (12.1 ± 1.1%). With calcium and vitamin D supplementation, the proportionate increase in urinary calcium/creatinine excretion was lower in smokers (44 ± 12%) than nonsmokers (79 ± 9%, p < 0.05). These results suggest that smoking accelerates bone loss from the femoral neck and total body in the elderly and that less efficient calcium absorption may be one contributing factor.

Evaluation of Scatter Correction using a Single Isotope for Simultaneous Emission and Transmission Data

Photon scatter is one of the most important factors degrading the quantitative accuracy of SPECT images. Many scatter correction methods have been proposed. The single isotope method was proposed by us. We evaluate the scatter correction method of improving the quality of images by acquiring emission and transmission data simultaneously with single isotope scan. To evaluate the proposed scatter correction method, a contrast and linearity phantom was studied. Four female patients with fibromyalgia (FM) syndrome and four with chronic back pain (BP) were imaged. Grey-to-cerebellum (G/C) and grey-to-white matter (G/W) ratios were determined by one skilled operator for 12 regions of interest (ROIs) in each subject. The linearity of activity response was improved after the scatter correction (r = 0.999). The y-intercept value of the regression line was 0.036 (p < 0.0001) after scatter correction and the slope was 0.954. Pairwise correlation indicated the agreement between nonscatter corrected and scatter corrected images. Reconstructed slices before and after scatter connection demonstrate a good correlation in the quantitative accuracy of radionuclide concentration. G/C values have significant correlation coefficients between original and corrected data. The transaxial images of human brain studies show that the scatter correction using single isotope in simultaneous transmission and emission tomography provides a good scatter compensation. The contrasts were increased on all 12 ROIs. The scatter compensation enhanced details of physiological lesions.

Prestorage white cell reduction in saline-adenine-glucose-mannitol red cells by use of an integral filter: evaluation of storage values and invivo recovery.

Prestorage white cell (WBC) reduction in blood components may decrease the incidence of adverse reactions and improve component quality. A bottom-and-top system with an integral third-generation WBC-reduction filter has been studied. Whole blood was collected from 30 healthy donors: from 20 by using a blood container system with an integral filter and from 10 controls by using a standard blood container system. Ten test units were buffy coat-depleted, stored for 72 hours at 4 degrees C, and then filtered, while an additional 10 test units were buffy coat-depleted and filtered at room temperature within 8 hours of collection. All units were stored at 4 degrees C for 42 days and sampled weekly. The mean WBC content of the 72-hour, 4 degrees C units was 0.33 x 10(6), that of the room-temperature units was 2.6 x 10(6), and that of the buffy coat-depleted controls was 460 x 10(6) (p < 0.0005). No significant differences were found among lactate, glucose, sodium, potassium, and plasma hemoglobin levels in the three groups. ATP and 2,3 DPG levels were significantly better preserved in control units than in 72-hour, 4 degrees C units (p = 0.016 and p = 0.032, respectively), but not better than in the room-temperature units. Significant differences were observed between pH values in filtered units and both groups of test units (p = 0.016). In biologic terms however, these differences were small. Red cells from an additional eight healthy volunteer donors were processed by an 8-hour room-temperature method and stored for 35 days. Studies in vivo 24-hour recovery of autologous red cells were performed by transfusing a radiolabeled (51Cr plus 131I-albumin) aliquot after 35 days' storage. Good recovery (mean > 80%) was found by both the single- and double-isotope-label methods. Recovery was significantly greater when calculated by the single-isotope method (p = 0.02). The combination of buffy coat removal and filtration in the blood container system with an integral filter achieved effective WBC reduction (> or = 3 log10 reduction from whole blood) without biologically significant detriment to in vitro or in vivo storage values.

Correction of glucose carbon recycling for the determination of ‘true’ hepatic glucose production rates by (1-13C1)glucose

Hepatic glucose production (HGP) and glucose carbon recycling are traditionally estimated by the combined use of hydrogen and carbon-labeled glucose tracers. A single-isotope method such as that of Reichard et al. for the determination of HGP and glucose carbon recycling requires the determination of activities in different glucose carbons by chemical degradation. Since the 13C content in the glucose carbon skeleton can be determined from mass fragmentography, the use of 13C-labeled glucose and mass fragmentography can provide a single-isotope method for the quantification of the recycled carbons. Correction for the recycling makes it possible to determine the true HGP. In this study, (1-13C1)glucose and mass fragmentography were used for the determination of HGP and glucose carbon recycling in six colon cancer patients. Molar enrichment of the molecular ion (m/z 328 cluster of glucose aldonitrile pentaacetate) was used to determine 'uncorrected' HGP, which was 1.93 +/- 0.11 mg kg-1 min-1 (mean +/- s.e.m.). The difference in molar enrichment of the molecular ion C1-C6 (m/z 328) and the ion corresponding to C1-C4 fragment (m/z 242) was used to determine the contribution of recycled label carbon. After this correction, the 'corrected' HGP was 2.04 +/- 0.12 mg kg-1 min-1, which is not significantly different from the 'true' HGP rate of 2.05 +/- 0.15 mg kg-1 min-1 determined by using (6-3H)glucose. HGP determined from the enrichment of the molecular ion C1-C6 underestimates true HGP, as expected. The corrected HGPs correlate well with those from 6-3H method (r = 0.86, y = 1.06x - 0.12; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of two-pool model for in vivo ketone body kinetics

Previous studies have indicated that simultaneous infusions of two ketone body tracers ([13C]acetoacetate and [14C]beta-hydroxybutyrate) provide accurate estimates of exogenous ketone body inflow when an open two-pool model is employed. In the present studies, net hepatic ketone body production was determined from surgically placed arterial, portal venous, and hepatic venous catheters in conscious diabetic (n = 6) and 4-day fasted (n = 7) dogs. [13C]acetoacetate and [14C]beta-hydroxybutyrate were infused simultaneously, and ketone body production was calculated from either acetoacetate (AcAc) single-isotope data, beta-hydroxybutyrate (beta-OHB) single-isotope data, the sum of individual fluxes, or the two-pool model. In fasted animals, both the AcAc single-isotope calculation and the sum of individual fluxes overestimated net hepatic production by approximately 50% (P less than 0.05), whereas the beta-OHB single-isotope calculation and the two-pool model gave accurate estimates. In the diabetic animals, the beta-OHB single-isotope calculation underestimated net hepatic production by approximately 30% (P less than 0.05). The sum of individual fluxes overestimated net hepatic production by approximately 46% (P less than 0.05), whereas both the AcAc single-isotope calculation and the two-pool model gave accurate estimates. In conclusion, single-isotope methods give erroneous estimates of net hepatic production of ketone bodies. In contrast, a two-pool model provided an accurate estimate of net hepatic production and thus appears to be suitable for determination of ketone body kinetics in humans.

Measurement of steady state protein degradation in cultured human muscle cells

Double label techniques for measurement of protein turnover in cultured cells are described. In the isotope withdrawal method protein in cultured muscle is labeled with two isotopes of the same amino acid for 24 to 100 h, followed by exposure to fresh medium containing one isotope only at the same specific activity for an additional 24 to 48 h. In the isotope addition method the order of addition of single and double-labeled media is reversed. After incubation the ratio of the two isotopes in the cell protein is a function of the incubation time and the degradation rate constant K d ; K d can readily be calculated using a graphical or iterative method. In mixed cultures of human muscle with initial incubation ranging 24 to 159 h, the K d 's obtained from various incubation times were similar. Both the isotope withdrawal and the isotope addition methods gave a K d value of 0.018 h −1 similar to values obtained by two different single isotope methods which monitor the appearance of free isotope in the medium of previously labeled cells. There were no differences of K d values obtained in cultures of muscle from normal patients and those with denervation, inflammatory myopathies, or nonspecific myopathic biopsy changes. When proteins were separated by gel electrophoresis, those of molecular weight greater than 60,000 had higher average K d values as compared to lower molecular weight proteins. The double isotope labeling method has the advantage of being easily applied to cultures with small numbers of cells and is potentially useful in obtaining the degradation rates of individual cellular proteins. The major disadvantages are (1) in their present form the methods can be used only in steady state cultures and (2) they require rather long (24 h) labeling times.

A single isotope method of post-prandial duodenogastric reflux assessment using 99Tcm-labelled IDA in patients with gallstones.

A technique for the quantitative assessment of post-prandial duodenogastric bile reflux is described using a single isotopes 99Tcm and a single-channel large-field gamma camera with a data processing system. The stomach is localised with pertechnetate prior to IDA administration and duodenogastric reflux is calculated as the percentage of hepatic IDA output reaching the stomach after correction for background activity and hepatic overlap. The technique has been validated, and used to study reflux in 25 patients with gallstones and in 10 control patients. Gall bladder function was assessed with an oral cholecystogram. Marked reflux (greater than 7%) occurred in 5 out 9 patients with a non-functioning gall bladder but in no controls and in none of 16 patients with gallstones in a functioning gall bladder. When patients were studied again after cholecystectomy, 2 patients with normal functioning gall bladders had developed marked reflux while those with preoperative reflux continued to reflux after cholecystectomy. Symptoms of gallstone dyspepsia before operation were more severe in those with marked reflux than those without. Surgery improved these symptoms even in those who continued to reflux after operation.

Simultaneous measurement of local glucose utilization and blood flow in the rat brain: an autoradiographic method using two tracers labeled with carbon-14.

A simplified technique that uses two radionuclide tracers has been devised to measure local cerebral glucose utilization (ICGU) and local cerebral blood flow (ICBF) in the same rat. The method employs [14C]-2-deoxyglucose and [14C]iodoantipyrine to produce an autoradiogram before and another after extraction into chloroform of the [14C]iodoantipyrine from the brain sections. The chloroform-extracted autoradiogram yields ICGU, and the difference in tissue carbon-14 concentration between the two autoradiograms permits calculation of ICBF. The double-isotope technique provides values of ICGU and ICBF that are statistically indistinguishable from those derived from conventional single-isotope methods.

Synthesis of myelin, particulate, and soluble protein subfractions of rat sciatic nerve during the early stage of Wallerian degeneration: a comparison of metabolic studies using double and single isotope methods and recovery.

The recovery, electrophoretic composition and synthesis of the myelin, particulate protein and soluble protein subfractions of rat sciatic nerve were compared in normal, sham-operated, and degenerating rat sciatic nerve at one, three and five days after neurotomy. Both single and double isotope methods were used to measure changes in synthesis in vitro and double isotope methods were used in vivo. The wet weights of nerves undergoing Wallerian degeneration for 5 days increased by 40 percent compared to normal and sham-operated nerves. The recovery, specific radioactivity, and synthesis of the myelin was reduced. The effect on myelin protein synthesis was similar in vitro and in vivo. The myelin loss was relatively constant in amount (30-40 microgram) regardless of differences in nerve sizes of young and old rats, consequently the percentage of myelin loss was inversely proportional to nerve size. The recovery of particulate protein increased, its rate of synthesis remained unchanged, and accordingly the specific radioactivity was decreased. The recovery, specific radioactivity, and the rate of synthesis of the soluble protein fraction were all elevated. The protein composition of the three fractions, as analyzed qualitatively by polyacrylamide disc gel electrophoresis, remained essentially unchanged through five days of degeneration. With regard to comparisons of the single and double isotope methods, results shows that the latter are more ideally suited to measuring changes in synthesis during the non-steady state conditions that are characteristics of rapid degeneration.

A single isotope method for the measurement of the volume of the mitochondrial inner compartment

A method is described to measure the volume of the inner mitochrondrial compartment using [ 51Cr]EDTA as an extracompartmental space marker and gravimetry to determine the total H 2O content and the weight of the mitochondria.

Dual-isotope separation technique for radioassay of Cobalamin

Cobalamin is assayed by a dual-isotope separation method using sodium [ 125I]iothalamate as a marker. Two systems are used: one in which the incompletely-separated bound fraction is counted and compared with the single-isotope method in which the bound fraction is separated by washing (Phadebas radiosorbent assay); and one in which an aliquot of the free fraction is counted. In the dual-isotope method counting bound fractions, about 97% of the supernatant is removed by pouring from silicone fluid separators. The results for serum samples obtained using dual- and single-isotope methods were similar (between run coefficients of variation 5–7%). Experimental errors were smaller in the dual-isotope method. A factor in the kit standards, presumably the absence of proteins, was found to affect the separation technique, resulting in relatively large experimental errors for standards in the single-isotope method. Washing the solid phase in the single-isotope method apparently resulted in a loss of bound isotope. In the dual-isotope method counting free fractions reasonable precision was obtained (coefficient of variation of serum samples 6.6%) even though only about 56% of the supernatant (free fraction) was counted.