Retinol isotope dilution accurately predicts liver reserves in piglets but overestimates reserves in lactating sows.

IMPACT STATEMENT: Vitamin A (VA) deficiency and hypervitaminosis A have been reported in groups of people worldwide. Conventional biomarkers of VA deficiency (e.g. serum retinol concentration, dose response tests) are not able to distinguish between sufficiency and hypervitaminosis A. Retinol isotope dilution (RID) predictions of VA status have been validated in humans and animal models from deficiency through toxicity; however, RID during life stages with unique issues related to isotopic tracing, such as infancy and lactation, requires further evaluation. This study investigated RID in piglets and lactating sows as models for human infants and women. In piglets, RID successfully determined VA deficiency (confirmed with liver analysis), and that the tracer mixes quickly. Conversely, in lactating sows, although serum and milk enrichments were similar, traditional RID equations overestimated VA stores, likely due to losses of tracer and higher extrahepatic VA storage than predictions. These data inform researchers about the challenges of using RID during lactation.

Phosphorus kinetics in lambs experimentally infected with Trichostrongylus colubriformis with the use of 32P.

This study was conducted to evaluate the metabolism and phosphorus (P) kinetics in lambs experimentally infected with Trichostrongylus colubriformis using the isotope dilution technique and modelling. Fifteen male lambs (21.1 ±â€¯1.50 kg) of the Santa Inês hair breed of approximately six months old, distributed in the treatments infected (I, n = 8) and control (C, n = 7) were used. The infected lambs received serial infections with 5000 T. colubriformis larvae, three times per week, for 3 weeks (45 000 T. colubriformis total larvae). After 66 days of the last inoculation of infective larvae, 6.6 MBq of 32P were injected in each lamb to evaluate the P kinetics. Blood, faeces and urine samples were collected in the following seven days and the slaughter of lambs were carried out on the last day in order to collect bone and soft tissues (Liver, kidney, heart and muscle) samples. To analyse P flows, the biomathematical model with four compartments (C1 - gastrointestinal tract, C2 - plasma, C3 - bone and C4 - soft tissue) was used. Similar P intake (VI) between treatments (C and I) was verified. Lower absorption of endogenous (Vaf) and dietary P (Vaa), as well as, lower amount of endogenous P (from saliva) that reaches the gastrointestinal tract (VIT), consequently, higher excretion of dietary P (VFD) were verified in infected lambs (P < 0.1). Additionally, in infected lambs, the P bioavailability was lower compared to control lambs. With the lower absorption (VaT) of P in infected lambs, there was, consequently, lower distribution to bones and soft tissues (VeD2) and lower P deposition in the bones (VO+D). It was concluded that P metabolism of lambs infected with T. colubriformis was altered, with reduced intestinal absorption and bioavailability, increased faecal loss and reduced P flow to the bone.

Evaluation of electrical broad bandwidth impedance spectroscopy as a tool for body composition measurement in cows in comparison with body measurements and the deuterium oxide dilution method.

Body fatness and degree of body fat mobilization in cows vary enormously during their reproduction cycle and influence energy partitioning and metabolic adaptation. The objective of the study was to test bioelectrical impedance spectroscopy (BIS) as a method for predicting fat depot mass (FDM), in living cows. The FDM is defined as the sum of subcutaneous, omental, mesenteric, retroperitoneal, and carcass fat mass. Bioelectrical impedance spectroscopy is compared with the prediction of FDM from the deuterium oxide (DO) dilution method and from body conformation measurements. Charolais × Holstein Friesian (HF; = 18; 30 d in milk) crossbred cows and 2 HF (lactating and nonlactating) cows were assessed by body conformation measurements, BIS, and the DO dilution method. The BCS of cows was a mean of 3.68 (SE 0.64). For the DO dilution method, a bolus of 0.23 g/kg BW DO (60 atom%) was intravenously injected and deuterium (D) enrichment was analyzed in plasma and whey by stabile isotope mass spectrometry, and total body water content was calculated. Impedance measurement was performed using a 4-electrode interface and time domain-based measurement system consisting of a voltage/current converter for applying current stimulus and an amplifier for monitoring voltage across the sensor electrodes. For the BIS, we used complex impedances over three frequency decades that delivers information on intra- and extracellular water and capacity of cell membranes. Impedance data (resistance of extra- and intracellular space, cell membrane capacity, and phase angle) were extracted 1) by simple curve fit to extract the resistance at direct current and high frequency and 2) by using an electrical equivalent circuit. Cows were slaughtered 7 d after BIS and D enrichment measurements and dissected for the measurement of FDM. Multiple linear regression analyses were performed to predict FDM based on data obtained from body conformation measurements, BIS, and D enrichment, and applied methods were evaluated by cross-validation. The FDM varied widely between cows and was correlated to D enrichment in plasma ( = 0.91, < 0.05). Prediction of FDM by body size measurements was less precise ( = 0.84), but FDM prediction was more accurate using D enrichment in plasma ( = 0.90) and BIS ( = 0.99) data. Therefore, both BIS and D enrichment analysis resulted in similarly good predictions of FDM in cows, and we conclude that BIS could have the potential to predict FDM in dairy cows from 40 to 380 kg.

Evaluation of the deuterium dilution method to estimate body composition in the barnacle goose: accuracy and minimum equilibration time.

We examined body composition in barnacle geese (Branta leucopsis) by proximate carcass analysis and by deuterium isotope dilution. We studied the effect of isotope equilibration time on the accuracy of total body water (TBW) estimates and evaluated models to predict fat-free mass (FFM) and fat mass (FM) from different measurements varying in their level of invasiveness. Deuterium enrichment determined at 45, 90, and 180 min after isotope injection did not differ significantly. At all sampling intervals, isotope dilution spaces (TBW(d)) consistently overestimated body water determined by carcass analysis (TBW(c)). However, variance in the deviation from actual TBW was higher at the 45-min sampling interval, whereas variability was the same at 90 and 180 min, indicating that 90 min is sufficient time to allow for adequate equilibration. At 90 min equilibration time, deuterium isotope dilution overestimated TBW(c) by 7.1% +/= 2.6% (P < 0.001, paired t-test, n=20). This overestimate was consistent over the range of TBW studied, and TBW(c) could thus be predicted from TBW(d) (r2=0.976, P<0.001). Variation in TBW(c) and TBW(d) explained, respectively, 99% and 98% of the variation in FFM. FM could be predicted with a relative error of ca. 10% from TBW estimates in combination with body mass (BM). In contrast, BM and external body measurements allowed only poor prediction. Abdominal fat fresh mass was highly correlated to total FM and, if the carcass is available, allows simple means of fat prediction without dissecting the entire specimen.

Comparison of various methods for estimating body fat in dogs.

Obesity is considered one of the most common forms of malnutrition occurring in dogs. Laboratory methods of evaluation of body composition in live dogs have included dual-energy X-ray absorptiometry (DEXA) and deuterium oxide (D(2)O) dilution. Clinical methods of evaluation include assigning a body condition score (BCS) based on visual observation, palpation, and morphometric measurements. This study used these four methods to evaluate 23 healthy, adult, client-owned dogs. Good correlation (coefficient of determination [r(2)]=0.78) was found between measurements of percent body fat (%BF) determined by the D(2)O dilution method and the DEXA scan. Percent body fat can also be estimated using BCS (r(2)=0.92 comparison with DEXA) or by using morphometric measurements with simple calculations (r(2)=0.92 comparison with DEXA).

Prediction of body composition of live and post-mortem red foxes.

A series of measurements (lengths, circumferences, skinfolds, masses and resistance) was taken on 29 red foxes (Vulpes vulpes) of both sexes before and after their death during the autumns of 1994 and 1995. Body composition of each carcass was determined by chemical analysis of homogenized samples of viscera, carcass and skin. Eight multiple regression models were then developed to predict body water, fat, protein, and mineral mass using body measurements as independent variables taken on live or dead animals. All final models were highly significant (P < 0.0001) and included three or four explanatory variables. Adjusted coefficients of determination varied between 0.95 for water mass and 0.81 for mineral mass. The models cover a wide range of conditions as percent body fat in the 29 samples varied between 1.1 and 28.4%. Our models can serve for management or research purposes with live or dead red foxes as they are quick, inexpensive and nondestructive.

Validation of predictive equations for use of deuterium oxide dilution to determine body composition of dogs.

OBJECTIVE: To validate equations predicting body composition of dogs, using deuterium oxide dilution. ANIMALS: 38 female and 37 male dogs selected by defined body weight and body condition criteria. PROCEDURE: Measured equilibrated deuterium concentration in serum after i.v. administration of isotope was used to determine isotope space and predict body water, fat, nitrogen, and ash contents determined from analysis of homogenized carcass. Equations predicting body composition were derived, using regression analysis, and were validated, using data-splitting techniques. RESULTS: Deuterium space (in kilograms) overestimated kilograms of body water content by mean 21.8% +/- SD of 4.18%. Regression equations were derived and validated to predict kilograms of body water and ash from kilograms of deuterium space, and proportion of body water, fat, nitrogen, and ash from proportion of deuterium space. Coefficients of determination (r2) and means of standard errors of estimating new values (SEE) were, respectively, 0.993 and 0.4 kg for body water content, and 0.942 and 0.08 kg for ash content. For proportions, SEE were 2.0, 2.7, 0.1, and 0.4% for body water, fat, nitrogen, and ash, respectively. Two factors, time for isotope equilibration and whether samples were processed by vacuum sublimation, improved SEE for proportion of body water from 2.0 to 1.3% and of fat from 2.7 to 1.8%. Equations predicting absolute quantities of fat and nitrogen could be derived but not validated. CONCLUSIONS: Deuterium dilution can be used to predict body composition of dogs with precision equal to that determined for other species.

Comparison of dual-energy x-ray absorptiometry and measurement of total body water content by deuterium oxide dilution for estimating body composition in dogs.

OBJECTIVE: To compare percentage of body fat (%BF) estimates from dual-energy x-ray absorptiometry (DEXA) with those derived from total body water (TBW) determination by deuterium oxide (D2O) dilution. ANIMALS: 31 healthy, adult, purebred dogs of various ages and breeds (body weight, 15 to 39 kg). PROCEDURE: The TBW was measured by D2O dilution and subsequent analysis via nuclear magnetic resonance (NMR). Blood was collected before and 2 hours after IV administration of 275 mg of D2O/kg of body weight. Plasma was separated and stored at -30 C until analysis by deuterium NMR. The DEXA scans were obtained immediately after blood collection from dogs under general anesthesia. RESULTS: Measurements of %BF by DEXA averaged 15.8% higher than calculated estimates of TBW content by D2O dilution. The linear regression of %BF by TBW content on %BF by DEXA had a slope of 1.04 and a correlation coefficient of 0.84, indicating excellent relative agreement between methods despite the significant difference in absolute agreement between the 2 methods. The average difference between methods did not differ by breed, sex, body condition score, body weight, or %BF, as measured by DEXA. CONCLUSION: Comparability of our data with those of previous studies suggest that DEXA is useful for in vivo estimation of body composition in healthy dogs. Body fat estimated by D2O dilution will be less than that determined by DEXA, despite excellent relative agreement between methods.

Measurement of total body water content in horses, using deuterium oxide dilution.

OBJECTIVE: To measure total body water (TBW) content in horses, using deuterium oxide (D2O) dilution. ANIMALS: Six 8- to 10-year-old healthy untrained mixed-breed horses, weighing (mean +/-SD) 503.4 +/- 64.0 kg. PROCEDURE: After a 12-hour nonfeeding period, 6 horses were given D2O (0.14 g/kg of body weight) via nasogastric tube. Blood samples were collected from a preplaced indwelling jugular vein catheter prior to and 1 to 8, 10, 12, 14, and 24 hours after administration of D2O. Blood samples were centrifuged immediately, and plasma was collected and stored at -70 C until analysis. The D2O content in plasma was measured by zinc reduction to deuterium gas. The resulting gas was measured, using an isotope ratio mass spectrometer. RESULTS: Deuterium oxide was rapidly absorbed from the gastrointestinal tract of all horses, and reached peak (mean +/- SD) plasma concentration (1,454.4 +/- 163 delta D/ml or parts/thousand) 1 hour after administration. Plasma concentration decreased slowly during the next 2 to 3 hours, then remained statistically constant from 2 to 5 hours (early plateau phase) and 3 to 7 hours (late plateau phase) after administration. Mean +/- SEM TBW content was 623.0 +/- 2.2 ml/kg (62.3% of body weight) for the early plateau phase and 630.3 +/- 2.2 ml/kg (63.0% of body weight) for the late plateau phase. CONCLUSION: Deuterium oxide dilution appears to be of value for measurement of TBW content in horses, and has a 4-hour plateau effect. Equilibration of D2O with large intestinal water may be the reason for the prolonged equilibrium time and plateau effect seen in these horses. CLINICAL RELEVANCE: Deuterium oxide appears safe and efficacious for determining TBW content in horses and may be helpful for determining changes in TBW content during exercise and disease.

Vitamin B12 levels in ewe colostrum and milk and in lamb serum.

Vitamin B12 was measured in samples of normal ovine colostrum and milk and in sera of lambs and ewes taken at various stages of lactation, from lambing up to 16 d. Colostrum obtained within 24 h of lambing contained high concentrations of vitamin B12, but within a few days the levels fell. The lambs received high levels of vitamin B12 from their dams' colostrum but a parallel decrease of vitamin B12 levels in lambs' serum and ewes' milk was subsequently observed. Milk vitamin B12 was analysed by the microbiological method (Lactobacillus leichmannii, ATCC 7830) and a radioisotope dilution (RID) technique. Milk vitamin B12 concentrations were greater with the microbiological technique compared with the RID method. The radioassay method has been shown to be as reliable as the microbiological method for measuring vitamin B12 in ovine milk except for very low levels.

Contribution of cobalamin analogues to plasma vitamin B12 concentrations in cattle.

Plasma vitamin B12 concentrations in cattle were analysed by a radioisotope dilution assay using pig intrinsic factor and a microbiological assay using Euglena gracilis. Both assays provided similar results for samples of cattle plasma containing vitamin B12 concentrations ranging from 0.07 to 3.60 micrograms litre-1 (r = 0.95, P less than 0.001). The addition of excess cobinamide in the radioisotope dilution assay to block non-specific binding in the intrinsic factor preparation due to the presence of R-type binders, was used to determine the presence of cobalamin analogues. Cobalamin analogues accounted for up to 50 per cent of the total vitamin B12 concentration in samples of plasma from cows but were virtually undetectable in plasma from sheep.

Plasma and erythrocyte volumes of newborn rabbits delivered after 30 days of gestation.

The plasma and erythrocyte volumes of 18 rabbit fetuses delivered by caesarean section on day 30 of gestation were measured by a double labelling radioisotope technique after separation from the placenta. These late gestation fetuses were viable. A negative correlation was found between the bodyweight of the neonate and its blood volume expressed as a percentage of bodyweight. The equation of the linear regression line was Y = 92.3 - 3.9X. The haematocrit was 0.494 +/- 0.029 l/l.