Milk beverage base with lactose removed with ultrafiltration: Effect of fat and protein concentration on sensory and physical properties.

Our objectives were to determine the effect of fat (skim to whole milk) and protein (3.4%-10.5%) concentration on the sensory and physical properties of milk beverage base that had lactose and other low molecular components removed by ultrafiltration (UF). In experiment 1, a matrix of 16 treatments was produced to achieve 4 levels of lactose removal (0%, 30%, 70%, and 97%) at each of 4 fat levels (skim, 1%, 2%, and whole milk). In experiment 2, a matrix of 12 treatments was produced to achieve 4 levels of lactose removal (0%, 30%, 70%, and 97%) at each of 3 protein concentrations (3.4%, 6.5%, and 10.5% protein). Physical and sensory properties of these products were determined. Removal of >95% of milk lactose by UF required a diafiltration volume of approximately 3 times the milk volume. Lactose and low molecular weight solute removal increased whiteness across the range from skim to whole milk while decreasing viscosity and making milk flavor blander. In addition, lactose (and other low molecular weight solute) removal by UF decreased titratable acidity by more than 50% and increased milk pH at 20°C to >7.0. Future work on milk and milk-based beverages with lactose removed by UF needs to focus on interaction of the remaining milk solids with added flavorings, changing casein to whey protein ratio before removal of lactose by UF, and the effect of lactose and low molecular weight solute removal on heat stability, particularly for neutral-pH, shelf-stable milk-based beverages.

The ultrafiltration molecular weight cut-off has a limited effect on the concentration and protein profile during preparation of human milk protein concentrates.

Optimizing protein intake for very low birth weight (<1,500 g) infants is fundamental to prevent faltering postnatal growth with the potential association of impaired neurodevelopment. The protein content of human milk is not sufficient to support the growth of very low birth weight infants. To meet their elevated protein requirements, human milk is currently fortified using typically bovine milk-based protein isolates (>85% on a dry basis). However, these products have several limitations for use in this vulnerable population. To overcome the shortcomings of bovine milk-based protein supplement, a human milk protein concentrate (HMPC) was developed. In preliminary attempts using 10 kDa ultrafiltration (UF) membranes, it was not possible to reach the protein content of commercial protein isolates, presumably due to the retention of human milk oligosaccharides (HMO). Consequently, it was hypothesized that the use of a UF membrane with a higher molecular weight cut-off (50 kDa rather than 10 kDa) could improve the transmission of carbohydrates, including HMO, in the permeate, thus increasing the protein purity of the subsequent HMPC. The results showed that permeate fluxes during the concentration step were similar to either UF molecular weight cut-off, but the 50-kDa membrane had a higher permeate flux during the diafiltration sequence. However, it was not sufficient to increase the protein purity of the human milk retentate, as both membranes generated HMPC with similar protein contents of 48.8% (10 kDa) and 50% (50 kDa) on a dry basis. This result was related to the high retention of HMO, mainly during the concentration step, although the diafiltration step was efficient to decrease their content in the HMPC. As the major bioactive proteins (lactoferrin, lysozyme, bile salt stimulated lipase, and α1-antitrypsin) in human milk were detected in both HMPC, the 50-kDa membrane seems the most appropriate to the preparation of HMPC in terms of permeation flux values. However, improving the separation of HMO from proteins is essential to increase the protein purity of HMPC.

Effect of lactose standardization of milk using low-concentration factor ultrafiltration: Effect of reducing the lactose-to-casein ratio on the properties of milled-curd Cheddar cheese.

The pH of cheese is determined by the amount of lactose fermented and the buffering capacity of the cheese. The buffering capacity of cheese is largely determined by the protein contents of milk and cheese and the amount of insoluble calcium phosphate in the curd, which is related to the rate of acidification. The objective of this study was to standardize both the lactose and casein contents of milk to better control final pH and prevent the development of excessive acidity in Cheddar cheese. This approach involved the use of low-concentration factor ultrafiltration of milk to increase the casein content (∼5%), followed by the addition of water, ultrafiltration permeate, or both to the retentate to adjust the lactose content. We evaluated milks with 4 different lactose-to-casein ratios (L:CN): 1.8 (control milk), 1.4, 1.1, and 0.9. All cheesemilks had similar total casein (2.3%) and fat (3.4%) contents. These milks were used to make milled-curd Cheddar cheese, and we evaluated cheese composition, texture, functionality, and sensory properties over 9 mo of ripening. Cheeses made from milks with varying levels of L:CN had similar moisture, protein, fat, and salt contents, due to slight modifications during manufacture (i.e., cutting the gel at a smaller size than control) as well as control of acid development at critical steps (i.e., cutting the gel, whey drainage, salting). As expected, decreasing the L:CN led to cheeses with lower lactic acid, residual lactose, and insoluble Ca contents, as well as a substantial pH increase during cheese ripening in cheeses. The L:CN ratio had no significant effect on the levels of primary and secondary proteolysis. Texture profile analysis showed no significant differences in hardness values during ripening. Maximum loss tangent, an index of cheese meltability, was lower until 45 d for the L:CN 1.4 and 0.9 treatments, but after 45 d, all reduced L:CN cheeses had higher maximum loss tangent values than the control cheese (L:CN 1.8). Sensory analyses showed that cheeses made from milks with reduced L:CN contents had lower acidity, sourness, sulfury notes, and chewdown cohesiveness. Standardization of milk to a specific L:CN ratio, while maintaining a constant casein level in the milk, would allow Cheddar cheese manufacturers to have tighter control of pH and acidity.

Short communication: Effect of pH on the heat stability of reconstituted reduced calcium milk protein concentrate dispersions.

This study aimed to investigate the heat stability of dispersions from reconstituted reduced-calcium milk protein concentrate (RCMPC) with 80% protein or more. The tested RCMPC powders were produced from skim milk subjected to CO2 treatment before and during the process of ultrafiltration. The CO2 injection was controlled to obtain 0 (control, no CO2 injection), 20, 30, and 40% reduction in calcium levels in the RCMPC powders. The RCMPC powders were reconstituted to 10% (wt/wt) protein in deionized water. These dispersions were tested for heat stability in a rocking oil bath at 140°C at unadjusted, 6.5, 6.7, 6.9, and 7.1 pH. Calcium ion activity (CIA) and ionic strength measurements were carried out using a Ca ion-selective electrode and conductivity meter. Unadjusted pH of the dispersions varied from 6.8 in control to 5.96 in 40% RCMPC dispersions. The CIA of unadjusted dispersions ranged from 1.31 mM in control to 2.83 mM in 40% RCMPC. Heat stability, expressed as heat coagulation time (HCT) of unadjusted dispersions decreased as the level of Ca removal in powders increased (from 13.81 min in control to 0.46 min in 40% RCMPC) and was negatively correlated with the CIA of the dispersions. For control RCMPC dispersions, the minimum and maximum heat stability were observed at dispersion pH of 6.5 and 6.9, respectively, followed by a decrease at pH 7.1 (CIA was the lowest). Dispersions from 40% RCMPC and pH 7.1 had the maximum HCT of 30.94 min among all RCMPC dispersions at all pH values. From this study, it can be concluded that improved heat stability in high protein formulation beverages subjected to UHT processing could be achieved through calcium reduction in milk protein concentrates using CO2 injection.

A rapid method to quantify casein in fluid milk by front-face fluorescence spectroscopy combined with chemometrics.

Casein in fluid milk determines cheese yield and affects cheese quality. Traditional methods of measuring casein in milk involve lengthy sample preparations with labor-intensive nitrogen-based protein quantifications. The objective of this study was to quantify casein in fluid milk with different casein-to-crude-protein ratios using front-face fluorescence spectroscopy (FFFS) and chemometrics. We constructed calibration samples by mixing microfiltration and ultrafiltration retentate and permeate in different ratios to obtain different casein concentrations and casein-to-crude-protein ratios. We developed partial least squares regression and elastic net regression models for casein prediction in fluid milk using FFFS tryptophan emission spectra and reference casein contents. We used a set of 20 validation samples (including raw, skim, and ultrafiltered milk) to optimize and validate model performance. We externally tested another independent set of 20 test samples (including raw, skim, and ultrafiltered milk) by root mean square error of prediction (RMSEP), residual prediction deviation (RPD), and relative prediction error (RPE). The RMSEP for casein content quantification in raw, skim, and ultrafiltered milk ranged from 0.12 to 0.13%, and the RPD ranged from 3.2 to 3.4. The externally validated error of prediction was comparable to the existing rapid method and showed practical model performance for quality-control purposes. This FFFS-based method can be implemented as a routine quality-control tool in the dairy industry, providing rapid quantification of casein content in fluid milk intended for cheese manufacturing.

A comparison of the heat stability of fresh milk protein concentrates obtained by microfiltration, ultrafiltration and diafiltration.

The objective of this work was to evaluate the impact of changes during membrane filtration on the heat stability of milk protein concentrates. Dairy protein concentrates have been widely employed in high protein drinks formulations and their stability to heat treatment is critical to ensure quality of the final product. Pasteurized milk was concentrated three-fold by membrane filtration, and the ionic composition was modified by addition of water or permeate from filtration (diafiltration). Diafiltration with water did not affect the apparent diameter of the casein micelles, but had a positive effect on heat coagulation time (HCT), which was significantly longer (50 min), compared to the non diafiltered concentrates (about 30 min). UHT treatments increased the particle size of the casein micelles, as well as the turbidity of retentates. Differences between samples with and without diafiltration were confirmed throughout further analysis of the protein composition of the unsedimentable fraction, highlighting the importance of soluble protein composition on the processing functionality of milk concentrates.

Short communication: Use of an ultrafiltration device in gland cistern for continuous sampling of healthy and mastitic quarters of lactating cattle for pharmacokinetic modeling.

Pharmacokinetic studies of the drugs in the milk are often limited due to infrequent sampling associated with milking. Alternatively, frequent sample collection with repeated milking may increase drug elimination. The objective of this study was to determine the feasibility of continuously sampling the udder using ultrafiltration. An ultrafiltration probe was placed into the gland cisterns through mammary parenchyma of normal and mastitic quarters of 6 mature mid-lactation Jersey cows with naturally occurring subclinical mastitis. An ultrafiltration probe was secured to the caudal or lateral aspect of the udder depending on the quarter being sampled. The timed interval samples were collected at 0, 2, 4, 6, 8, 12, 18, 24, 28, 32, 36, 48, 60, 72, 84, and 96 h after drug administration. Plasma samples were collected at the same time points. Each cow received 2.2 mg/kg of flunixin intravenously before milking at time 0. All cows were routinely milked by machine every 12 h. Flunixin concentrations in plasma, whole milk, and milk ultrafiltrates were analyzed by use of ultra-high-performance liquid chromatography with mass spectrometric detection. We found no significant effects on the appearance of the milk or the ability to milk the cows after implantation of the ultrafiltration probes. The concentration of flunixin collected from the ultrafiltration probes in the mastitic quarters tended to be greater than that of the healthy quarters. We concluded that collection of ultrafiltration samples from the mammary gland of cows provides a viable means to continuously assess drug concentrations in the milk while continuing to milk the cow normally. This study demonstrates the utility of continuous sampling of milk via ultrafiltration for future pharmacokinetic studies in cattle.

Carprofen pharmacokinetics in plasma and in control and inflamed canine tissue fluid using in vivo ultrafiltration.

Measurement of unbound drug concentrations at their sites of action is necessary for accurate PK/PD modeling. The objective of this study was to determine the unbound concentration of carprofen in canine interstitial fluid (ISF) using in vivo ultrafiltration and to compare pharmacokinetic parameters of free carprofen concentrations between inflamed and control tissue sites. We hypothesized that active concentrations of carprofen would exhibit different dispositions in ISF between inflamed vs. normal tissues. Bilateral ultrafiltration probes were placed subcutaneously in six healthy Beagle dogs 12 h prior to induction of inflammation. Two milliliters of either 2% carrageenan or saline control was injected subcutaneously at each probe site, 12 h prior to intravenous carprofen (4 mg/kg) administration. Plasma and ISF samples were collected at regular intervals for 72 h, and carprofen concentrations were determined using HPLC. Prostaglandin E2 (PGE2 ) concentrations were quantified in ISF using ELISA. Unbound carprofen concentrations were higher in ISF compared with predicted unbound plasma drug concentrations. Concentrations were not significantly higher in inflamed ISF compared with control ISF. Compartmental modeling was used to generate pharmacokinetic parameter estimates, which were not significantly different between sites. Terminal half-life (T½) was longer in the ISF compared with plasma. PGE2 in ISF decreased following administration of carprofen. In vivo ultrafiltration is a reliable method to determine unbound carprofen in ISF, and that disposition of unbound drug into tissue is much higher than predicted from unbound drug concentration in plasma. However, concentrations and pharmacokinetic parameter estimates are not significantly different in inflamed vs. un-inflamed tissues.

Production of goat milk protein hydrolysate enriched in ACE-inhibitory peptides by ultrafiltration.

A global process for the production of goat milk hydrolysates enriched in angiotensin converting enzyme (ACE) inhibitory peptides was proposed. Firstly, the protein fractions (caseins and whey proteins) were separated by ultrafiltration through a 0·14 µm ceramic membrane. The casein fraction obtained in the retentate stream of the above filtration step was subsequently hydrolysed with a combination of subtilisin and trypsin. After 3 h of reaction, the hydrolysate produced presented an IC50 of 218·50 µg/ml, which represent a relatively high ACE inhibitory activity. Finally, this hydrolysate was filtered through a 50 kDa ceramic membrane until reaching a volume reduction factor of 3. The permeate produced presented an improvement of more than 30% in the ACE inhibitory activity. In contrast, the retentate was concentrated in larger and inactive peptides which led to a decrease of more than 80% in its inhibitory activity. The process suggested in this work was suitable to obtain a potent ACE inhibitory activity product able to be incorporated into food formulas intended to control or lower blood pressure. Moreover, the liquid product could be easily stabilised by spray dried if it would be necessary.

Platinum measurements in Dulbecco's phosphate-buffered saline are lower than in plasma and can be sampled with ultrafiltration probes in vitro.

OBJECTIVE: To compare the influence of fluid on carboplatin elution, and assess the feasibility of ultrafiltration (UF) probe sampling. SAMPLE: 20 samples of 5 mg carboplatin in 1.0 mL 30% poloxamer 407 eluting in Dulbecco's phosphate-buffered saline (DPBS) or canine plasma and 6 samples of UF probe sampling in 0.01 mg/mL carboplatin in DPBS or plasma. METHODS: Carboplatin-gel specimens in dialysis tubing (12- to 14-kDa pores) were placed in 100 mL of DPBS or canine plasma (37 °C and 600 rpm stirring) in a nonlidded and lidded experiment. Samples were collected in decreasing frequency for 96 hours. The 0.01-mg/mL carboplatin solutions in DPBS and plasma were sampled 6 times by UF probe (30-kDa pores) or direct aspiration. Platinum was measured using inductively coupled plasma mass spectrometry. RESULTS: High fluid evaporation was noted in the nonlidded but not the lidded experiment. A burst release was seen in plasma (first 2 hours) and DPBS (first 5 hours) with the highest hourly increase in the first hour in both DPBS (6,040 ppb/h) and plasma (2,612 ppb/h), with no further increase after the first 22 hours. Platinum content in the specimens was higher at 96 hours than the surrounding fluid. Higher platinum concentrations were measured by both direct and UF probe sampling in DPBS than in plasma. CLINICAL RELEVANCE: Platinum concentrations measured in DPBS were higher than in plasma, but elution patterns were similar. Ultrafiltration probes can be used to sample platinum in vitro and could be used in vivo to measure local unbound Pt tissue concentrations in local chemotherapy delivery.

Continuous sampling of healthy and mastitic quarters of lactating cattle by ultrafiltration after intramammary ceftiofur hydrochloride administration.

BACKGROUND: Pharmacological activity of intramammary drugs depends on adequate drug concentrations within the cistern, but sampling is often limited. Insight into the active drug concentration within the mammary cistern may assist in determining effective and appropriate therapeutic decisions for cows being treated for mastitis. OBJECTIVE: Evaluate the disposition of ceftiofur hydrochloride administered intramammary in diseased and nondiseased quarters. Whole milk and ultrafiltrate sampling techniques were compared. ANIMALS: Ten mature, late lactation Holstein (n = 9) and Jersey (n = 1) dairy cows (422-670 kg) with naturally occurring clinical mastitis, producing between 1.4 and 15.9 kg/day of milk. METHODS: Ultrafiltration probes were placed in both mastitic and healthy quarters. Each quarter was treated with 2 doses of 125 mg ceftiofur hydrochloride suspension, and whole milk and milk ultrafiltrate samples were collected. Ceftiofur concentrations in composite whole milk and milk ultrafiltrate were analyzed. RESULTS: The maximum concentration of ceftiofur was higher in ultrafiltrate samples, but no differences were identified in healthy or mastitic quarters. The use of ultrafiltration probes provides a novel technique for free drug concentrations within the mastitic and healthy bovine mammary gland. CONCLUSIONS AND CLINICAL IMPORTANCE: Significant inter- and intracow variability and lower daily milk weights may overestimate ceftiofur concentrations available within the cistern. The pharmacokinetic (PK) parameters reported in milk ultrafiltrate will help establish a link between the PK and the corresponding drug effect, potentially providing a meaningful rationale for the selection of a safe and effective dose in cows with mastitis.

Pharmacokinetic study on pradofloxacin in the dog - comparison of serum analysis, ultrafiltration and tissue sampling after oral administration.

BACKGROUND: Pradofloxacin, a newly developed 8-cyano-fluoroquinolone, show enhanced activity against Gram-positive organisms and anaerobes to treat canine and feline bacterial infections. The purpose of this cross-over study was to measure the unbound drug concentration of pradofloxacin in the interstitial fluid (ISF) using ultrafiltration and to compare the kinetics of pradofloxacin in serum, ISF and tissue using enrofloxacin as reference. RESULTS: After oral administration of enrofloxacin (5 mg/kg) and pradofloxacin (3 mg/kg and 6 mg/kg, respectively), serum collection and ultrafiltration in regular intervals over a period of 24 h were performed, followed by tissue sampling at the end of the third dosing protocol (pradofloxacin 6 mg/kg). Peak concentrations of pradofloxacin (3 mg/kg) were 1.55±0.31 µg/ml in the ISF and 1.85±0.23 µg/ml in serum and for pradofloxacin (6 mg/kg) 2.71±0.81 µg/kg in the ISF and 2.77±0.64 µg/kg in serum; both without a statistical difference between ISF and serum. Comparison between all sampling approaches showed no consistent pattern of statistical differences. CONCLUSIONS: Despite some technical shortcomings the ultrafiltration approach appears to be the most sensitive sampling technique to estimate pharmacokinetic values of pradofloxacin at the infection site. Pharmacokinetics - Pradofloxacin - Ultrafiltration - Dog - Oral Administration.

Noncasein nitrogen analysis of ultrafiltration and microfiltration retentate.

Previous research has suggested that the standard noncasein nitrogen (NCN) measurement method for milk overestimates the NCN content of microfiltration (MF) retentate. The objective of this study was to develop a modified method to more accurately measure the NCN content of ultrafiltration and MF retentate products. The standard method is based on precipitation of casein micelles at their isoelectric point (4.6) with acetic acid. In the standard method, a 10-mL milk sample and 75 mL of 38°C water are placed in a 100-mL volumetric flask. One milliliter of 10% acetic acid solution is added and the flask is incubated at 38°C for 10 min. Subsequently, 1 mL of 1N sodium acetate solution is added and mixed. After cooling the contents to 20°C, the flask is made up to 100mL with water, mixed, and then filtered (Whatman No. 1 filter paper). The N content of the filtrate is then determined by Kjeldahl analysis and referred to as NCN. A method was developed that used a 50-mL centrifugal tube instead of a volumetric flask. This modification facilitated measurement of the pH after addition of acetic acid. Subsequently, the sample was centrifuged (800×g at 25°C) for 10 min to facilitate filtration with a smaller pore size filter paper (Whatman no. 6). In this study, we evaluated the effect of pH after addition of 1% acetic acid and pH of the final filtrate on NCN analysis. Four pH levels after acetic acid addition (4.0, 4.2, 4.4, and 4.6) and 2 pH levels after sodium acetate addition (4.6 and 4.8) were evaluated. As the pH after acetic acid addition was increased from 4.0 to 4.6, the NCN content significantly decreased. Sodium dodecyl sulfate PAGE results also indicated that the casein fractions present in the filtrate were significantly decreased when the pH was increased from 4.0 to 4.6. The NCN content slightly decreased but the difference was not significant when the final pH of the filtrate was increased from 4.6 to 4.8. Subsequently, the NCN contents of several ultrafiltration and MF samples were determined using the standard method and modified method. The modified method gave significantly lower NCN values for most samples as compared with the standard method.

Effect of modified ultrafiltration on cytokines and hemoconcentration in dogs undergoing cardiopulmonary bypass.

Cardiac surgery using cardiopulmonary bypass (CPB) generates severe inflammatory reactions secondary to hemodilution and surgical stress. This study was conducted to evaluate whether modified ultrafiltration (MUF) could be performed safely and to clarify its effects during mitral valve repair in dogs in terms of hemodilution and the status of inflammatory cytokines. We retrospectively studied 38 dogs with mitral valve disease who underwent MUF immediately after mitral valve repair under CPB. To determine the effect of MUF, we measured the pre- and post-MUF blood dilution and blood cytokine levels. The levels of red blood cells, hematocrit (HCT), and albumin were significantly increased after MUF, whereas interleukin (IL)-6 levels were significantly increased from 24.3 (range 9.6-54.6) to 32.3 (15.9-65.1) pg/ml. The levels of IL-8 and IL-10 declined significantly after MUF, from 368.2 (246.1-669.4) and 45.4 (28.6-76.1) to 272.2 (174.1-414.4) and 28.8 (18.8-44.5) pg/ml, respectively. Our results demonstrated that MUF can be applied in dogs undergoing CPB and is effective in achieving hemoconcentration. Moreover, MUF may be useful for the removal of cytokines. Further studies are needed to validate these findings and clarify the effects of inflammatory cytokines after CPB.

Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae.

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P<0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P<0.001) 17h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus).

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the world's most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx. Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50x). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.

Fractionation of calcium and magnesium in equine serum.

OBJECTIVE: To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions. ANIMALS: 28 clinically normal horses. PROCEDURE: Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (microCa) and magnesium (microMg) fractions. Serum concentrations of iCa and iMg were measured in the ultrafiltrate by use of selective electrodes. Serum concentration of complexed calcium (cCa) or magnesium (cMg) was calculated by subtracting iCa or iMg from microCa or microMg, respectively. RESULTS: Mean +/-SE serum tCa concentration was 3.26 +/- 0.06 mmol/L. Calcium fractions were as follows: pCa, 1.55 +/- 0.03 mmol/L (47.4 +/- 0.9%); iCa, 1.58 +/- 0.03 mmol/L (48.5 +/- 0.7%); and cCa, 0.13 +/- 0.02 mmol/L (4.1 +/- 0.9%). Serum tMg concentration was 0.99 +/- 0.04 mmol/L. Magnesium fractions were as follows: pMg, 0.33 +/- 0.04 mmol/L (33.3 +/- 4.2%); iMg, 0.57 +/- 0.02 mmol/L (57.6 +/- 1.7%); and cMg, 0.09 +/- 0.02 mmol/L (9.1 +/- 1.9%). Refrigeration (4 degrees C) did not affect iCa values, whereas iMg declined by 8% after 120 hours. Neither iCa nor iMg was affected by freezing (-20 degrees C). CONCLUSIONS AND CLINICAL RELEVANCE: In equine serum, iMg is less stable than iCa; thus, when serum samples are not going to be analyzed promptly, freezing may be preferable to refrigeration for storage.

Fractionation of canine serum magnesium.

BACKGROUND: Serum total magnesium (tMg) consists of 3 fractions: ionized magnesium (iMg), protein-bound magnesium (pbMg), and complexed magnesium (cMg). Serum iMg may be measured by an ion-selective electrode, but determination of pbMg and cMg has not been attempted in dogs. OBJECTIVES: The objectives of this study were to assess the validity of a micropartition system to fractionate serum tMg and to establish reference intervals for pbMg, cMg, and iMg in clinically normal dogs using this method. METHODS: Serum samples from 10 clinically healthy dogs were fractionated using a micropartition system (Centrifree YM-30, Amicon Corp, Lexington, MA, USA). Serum tMg and iMg were measured in whole serum, and tMg was also measured in the ultrafiltrate. Concentration of cMg was obtained by the subtraction of iMg from tMg concentrations of the ultrafiltrate. Protein-bound Mg was calculated by subtracting the tMg concentration of the ultrafiltrate from the tMg concentration of whole serum. RESULTS: Results for pbMg and cMg using the micropartition system showed good reproducibility. Determination of tMg and iMg had acceptable inter- and intra-assay precision. Concentrations of iMg, cMg, and pbMg were 0.50 +/- 0.05 mmol/L, 0.05 +/- 0.04 mmol/L, and 0.24 +/- 0.04 mmol/L, representing 63%, 6%, and 31% of the tMg concentration, respectively. CONCLUSIONS: The micropartition system was a reproducible means to accurately assess cMg and pbMg concentrations in dogs.

Plasma pharmacokinetics and tissue fluid concentrations of meropenem after intravenous and subcutaneous administration in dogs.

OBJECTIVE: To estimate pharmacokinetic variables and measure tissue fluid concentrations of meropenem after IV and SC administration in dogs. ANIMALS: 6 healthy adult dogs. PROCEDURE: Dogs were administered a single dose of meropenem (20 mg/kg) IV and SC in a crossover design. To characterize the distribution of meropenem in dogs and to evaluate a unique tissue fluid collection method, an in vivo ultrafiltration device was used to collect interstitial fluid. Plasma, tissue fluid, and urine samples were analyzed by use of high-performance liquid chromatography. Protein binding was determined by use of an ultrafiltration device. RESULTS: Plasma data were analyzed by compartmental and noncompartmental pharmacokinetic methods. Mean +/- SD values for half-life, volume of distribution, and clearance after IV administration for plasma samples were 0.67 +/- 0.07 hours, 0.372 +/- 0.053 L/kg, and 6.53 +/- 1.51 mL/min/kg, respectively, and half-life for tissue fluid samples was 1.15 +/- 0.57 hours. Half-life after SC administration was 0.98 +/- 0.21 and 1.31 +/- 0.54 hours for plasma and tissue fluid, respectively. Protein binding was 11.87%, and bioavailability after SC administration was 84%. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of our data revealed that tissue fluid and plasma (unbound fraction) concentrations were similar. Because of the kinetic similarity of meropenem in the extravascular and vascular spaces, tissue fluid concentrations can be predicted from plasma concentrations. We concluded that a dosage of 8 mg/kg, SC, every 12 hours would achieve adequate tissue fluid and urine concentrations for susceptible bacteria with a minimum inhibitory concentration of 0.12 microg/mL.

Determination of calcium fractionation in dogs with chronic renal failure.

OBJECTIVE: To determine concentrations of calcium (total [tCa], ionized [iCa], protein-bound [pCa], and complexed [cCa]) in dogs with chronic renal failure (CRF). ANIMALS: 23 dogs with CRF. PROCEDURE: Serum calcium was fractionated by use of a micropartition system. Total calcium and iCa concentrations and pH were measured in unfractionated serum, and tCa concentration was measured in the ultrafiltrate. The pCa fraction was calculated by subtracting tCa of the ultrafiltrate from tCa concentration of unfractionated serum. The iCa concentration in unfractionated serum was subtracted from tCa concentration in the ultrafiltrate to determine the concentration of cCa. RESULTS: Concentrations of tCa, iCa, pCa, and cCa had wide ranges among dogs with CRF Dogs with significantly low tCa concentration (770 +/- 1.73 mg/dL) had cCa concentration (0.76 +/- 0.38 mg/dL) within reference range, whereas dogs with reference range to high tCa concentration (10.85 +/- 1.13 mg/dL) had significantly high cCa concentration (2.62 +/- 1.04 mg/dL). There was no significant difference in iCa or pCa concentrations between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of tCa, iCa, cCa, and pCa varied widely in dogs with CRF Overall, cCa concentration was high, although subpopulations differed in cCa and tCa concentrations. Differences in tCa concentration were primarily attributable to differences in cCa fraction.