The usefulness and limitations of hand-held refractometers in veterinary laboratory medicine: an historical and technical review

Abstract: Medical hand-held refractometers have been used in veterinary practice since their development in the 1960s. They have become ubiquitous for the measurement of protein and urine solute concentrations because of their rapidity of analysis, ease of use, and relatively low cost. Refraction of light offers advantages for the determination of solute concentrations because the measurement requires no chemical alteration of the specimen. Numerous authors have reported that the results of protein estimation by refractometry for domestic mammals correlate well with those obtained by the biuret method, although others have reported both higher and lower refractometric results compared with biuret results. Major discrepancies between biuret and refractometric results have been reported for avian samples. Some of the variation in reported results may be due to differences in design by refractometer manufacturers. Another possible source may be variation in the biuret reagent mixture and assay conditions. Refractometers also can be used to calculate serum water concentration. A table that converts index of refraction to serum water concentration can be used to convert electrolyte concentration from mmol/L of serum to mmol/L of serum water, a more accurate indicator of effective electrolyte concentration. Refractometers are especially useful for determining urine specific gravity on veterinary samples because they require relatively small sample volumes. Specific gravity continues to be the most common unit for reporting total solids concentration. Some solutes, such as acetone, may cause false increases in specific gravity by refractometry, as they increase refraction but are less dense than water.     

The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid

The aim of this study was to evaluate the influence of two commonly used anticoagulants (K3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K3EDTA, a solution of K3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K3EDTA. The solution of K3EDTA in distilled water only caused TP overestimation at high K3EDTA concentrations (>5 micromol/ml). By contrast, lithium heparin did not influence the refractometric values of TP. Neither anticoagulant modified TP values when measured by the biuret method. In conclusion, the use of K3EDTA as anticoagulant may result in a significant overestimation of TP values of peritoneal fluid samples measured by refractometry.     

Determination of chicken and turkey plasma and serum protein concentrations by refractometry and the biuret method

Plasma and serum protein concentrations were determined in chickens and turkeys by refractometry (with human and veterinary refractometers) and by the biuret method. Chicken and turkey serum protein values were significantly lower than respective plasma protein values according to both methods. Refractometer readings for both plasma and serum correlated closely with the results of the biuret test (r2 = 0.72 to 0.97). These findings indicate that plasma and serum protein values may be determined accurately in chickens and turkeys with a handheld refractometer.     

Comparison of refractometers and test endpoints in the measurement of serum protein concentration to assess passive transfer status in calves

OBJECTIVE: To evaluate 3 refractometers for detection of failure of passive transfer (FPT) of immunity in calves, and assess the effect of refractometric test endpoints on sensitivity, specificity, and proportion of calves classified correctly with regard to passive transfer status. DESIGN: Prospective study. ANIMALS: 90 calves. PROCEDURE: Blood samples were obtained from calves that were < 10 days old. Serum IgG concentration was determined by use of a radial immunodiffusion assay. Accuracy of 3 refractometers in the prediction of serum IgG concentration was determined by use of standard epidemiologic methods and a linear regression model. RESULTS: At a serum protein concentration test endpoint of 5.2 g/dL, sensitivity of each refractometer was 0.89 or 0.93, and specificity ranged from 0.80 to 0.91. For all refractometers, serum protein concentration test endpoints of 5.0 or 5.2 g/dL resulted in sensitivity > 0.80, specificity > 0.80, and proportion of calves classified correctly > 0.85. Serum protein concentrations equivalent to 1,000 mg of IgG/dL of serum were 4.9, 4.8, and 5.1 g/dL for the 3 refractometers. CONCLUSIONS AND CLINICAL RELEVANCE: The refractometers, including a nontemperature-compensating instrument, performed similarly in detection of FPT. Serum protein concentration test endpoints of 5.0 and 5.2 g/dL yielded accurate results in the assessment of adequacy of passive transfer; lower or higher test endpoints misclassified larger numbers of calves.     

Comparison of four methods for determination of total protein concentrations in pleural and peritoneal fluid from dogs

OBJECTIVE: To compare 4 techniques for determination of total protein concentrations in peritoneal and pleural effusions from dogs. SAMPLE POPULATION: 23 peritoneal and 12 pleural fluid samples from 35 dogs with various abnormalities. PROCEDURE: Samples were collected into tubes containing EDTA, centrifuged, and stored at -20 C until total protein concentrations were assessed. Protein concentration in each sample was determined by use of urine test strips, refractometry, and Bradford and biuret techniques. Accuracy of each method was determined, using dilutions of human control sera. RESULTS: There was good correlation among results of all quantitative procedures. Results of the biuret technique were more accurate than results of the Bradford assay. Refractometry underestimated protein concentration in samples with < 20 g of protein/L. Results of urine test strips correctly classified effusion samples into 2 groups on the basis of total protein concentrations less than or greater than 20 g/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results of any of these 4 techniques can be used to rapidly and efficiently differentiate peritoneal and pleural fluid from dogs into transudates and exudates on the basis of total protein concentration less than or greater than 20 g/L, respectively.     

Analysis of transtracheal aspirates and pleural fluid from clinically healthy llamas (Llama glama)

Seventeen clinically healthy adult llamas were used to study the characteristics of transtracheal aspirates (TTA) and pleural fluid samples. Results of complete blood counts, fibrinogen determination and thoracic radiographs were within normal limits prior to sampling. Cytologic evaluation of TTA revealed the majority of cells were vacuolated macrophages (60-100%), with 0-40% neutrophils, and fewer lymphocytes (0-1%), eosinophils (0-3%), and ciliated respiratory epithelial cells (0-10%). In TTA from 10 of 17 llamas, neither aerobic nor anaerobic bacteria were isolated. Bacteria isolated in pure culture from TTA were similar to isolates found in clinically healthy animals of other species, and included Acinetobacter sp., Staphylococcus sp. and Bacillus sp. Results (mean +/- SD) of pleural fluid analyses were: total nucleated cell count 576 +/- 361/microliter; specific gravity 1.0133 +/- 0.002; glucose concentration 135.1 +/- 9.02 mg/dL; and lactate concentration 2.95 +/- 1.34 mg/dL. Pleural fluid total protein concentrations determined by refractometry ranged from < 2.5 to 3.5 g/dL. The refractive index ranged from 1.3396 to 0.0013. In pleural fluid, small lymphocytes were the predominant cell type.     

Use of refractometry for determination of psittacine plasma protein concentration

Background: Previous studies have demonstrated both poor and good correlation of total protein concentrations in various avian species using refractometry and biuret methodologies. Objectives: The purpose of the current study was to compare these 2 techniques of total protein determination using plasma samples from several psittacine species and to determine the effect of cholesterol and other solutes on refractometry results. Methods: Total protein concentration in heparinized plasma samples without visible lipemia was analyzed by refractometry and an automated biuret method on a dry reagent analyzer (Ortho 250). Cholesterol, glucose, and uric acid concentrations were measured using the same analyzer. Results were compared using Deming regression analysis, Bland–Altman bias plots, and Spearman's rank correlation. Results: Correlation coefficients (r) for total protein results by refractometry and biuret methods were 0.49 in African grey parrots (n=28), 0.77 in Amazon parrots (20), 0.57 in cockatiels (20), 0.73 in cockatoos (36), 0.86 in conures (20), and 0.93 in macaws (38) (P≤.01). Cholesterol concentration, but not glucose or uric acid concentrations, was significantly correlated with total protein concentration obtained by refractometry in Amazon parrots, conures, and macaws (n=25 each, P<.05), and trended towards significance in African grey parrots and cockatoos (P=.06). Conclusions: Refractometry can be used to accurately measure total protein concentration in nonlipemic plasma samples from some psittacine species. Method and species‐specific reference intervals should be used in the interpretation of total protein values.     

Comparison of a digital and an optical analogue hand-held refractometer for the measurement of canine urine specific gravity

Urine specific gravity (USG) is used clinically as a measure of urine concentration, and is routinely assessed by refractometry. A comparison between optical analogue and digital refractometers for evaluation of canine urine has not been reported. The aim of this study was to compare a digital and an optical analogue hand-held refractometer for the measurement of canine USG, and to assess correlation with urine osmolality. Prospective study. Free-catch urine samples were collected from 285 hospitalised adult dogs, and paired USG readings were obtained with a digital and an optical analogue refractometer. In 50 dogs, urine osmolality was also measured using a freezing point depression osmometer. There was a small but statistically significant difference between the two refractometers (P<0.001), with the optical analogue refractometer reading higher than the digital refractometer (mean difference 0.0006, sd 0.0012). Paired refractometer measurements varied by <0.002 in 91.5 per cent of cases. The optical analogue and digital refractometer readings showed excellent correlation with osmolality (r=0.980 and r=0.977, respectively, P<0.001 in both cases). Despite statistical significance, the difference between the two refractometers is unlikely to be clinically significant. Both instruments provide an accurate assessment of USG in dogs.     

Relationship of protein concentration and water content of equine serum and plasma samples

A highly significant correlation between the water content and protein concentration of equine serum and plasma samples was demonstrated over a wide range of concentrations. A close correlation was also observed between protein concentration as estimated by refractometry and as determined by the biuret procedure for equine serum and plasma samples.     

Evaluation of assay procedures for prediction of passive transfer status in lambs

OBJECTIVE: To compare 4 assay procedures for prediction of passive transfer status in lambs. ANIMALS: Thirty-one 1-day-old Sardinian lambs. PROCEDURE: Serum IgG concentration was determined by use of single radial immunodiffusion. The following were determined: serum total protein concentration as measured by refractometry (ie, refractometry serum total protein concentration), serum total protein concentration as determined by the biuret method (ie, biuret method serum total protein concentration), serum gamma-globulin concentration as determined by serum protein electrophoresis, and serum gamma-glutamyltransferase (GGT) activity as measured by spectrophotometry. Accuracy of these assays for estimation of serum IgG concentration in 1-day-old lambs was established by use of linear regression analysis. RESULTS: Refractometry serum total protein concentration, biuret method serum total protein concentration, and serum gamma-globulin concentration were closely and linearly correlated with serum IgG concentration. The natural logarithm (ln) of serum GGT activity was closely and linearly correlated with serum IgG concentration (ln). Refractometry serum total protein concentration, biuret method serum total protein concentration, and gamma-globulin concentration accounted for approximately 85%, 91%, and 95% of the variation in serum IgG concentration, respectively. Serum GGT activity (ln) accounted for approximately 92% of the variation in serum IgG concentration (ln). CONCLUSIONS AND CLINICAL RELEVANCE: For prediction of passive transfer status in 1-day-old lambs, serum GGT activity or biuret method serum total protein concentration determination will allow for passive transfer monitoring program development. Immediate refractometry serum total protein concentration determination is beneficial in making timely management and treatment decisions. Serum gamma-globulin concentration determination can be used as a confirmatory test.     

Peritoneal fluid analysis in adult, nonpregnant Awassi sheep

BACKGROUND: To the authors' knowledge, there is no information in the literature about normal peritoneal fluid values in ovine species. OBJECTIVES: The purpose of the study reported here was to establish reference intervals for peritoneal fluid from clinically normal Awassi sheep and to compare the values to those in blood. METHODS: Peritoneal fluid and blood samples were collected into tubes containing EDTA, from 40 clinically healthy, nonpregnant, female Awassi sheep, aged 2 to 7 years. Total nucleated cell count (TNCC) was determined using an electronic cell counter. Total protein, albumin, urea, creatinine, and glucose concentrations and aspartate transaminase activity were analyzed using commercially available kits. RESULTS: TNCC (mean +/- SD) of peritoneal fluid was 1.1 +/- 0.87 X 10(3)/microl, with neutrophils (3.9%), lymphocytes (33.5%), macrophages/monocytes (61.2%), and eosinophils (1.4%). Biochemical results in peritoneal fluid were: total protein, 1.7 +/- 0.74 g/dL; albumin, 1.0 +/- 0.04 g/dL; urea, 12.6 +/- 3.95 mg/dL; creatinine, 0.6 +/- 0.19 mg/dL; glucose, 54.8 +/- 6.11 mg/dL; and aspartate transaminase, 23.5 +/- 8.82 U/L. Eosinophil percentage and creatinine concentration did not differ significantly from blood values. CONCLUSION: Baseline values for cytologic and biochemical parameters in peritoneal fluid of Awassi sheep, with comparison to blood, have been generated. Such data may be applicable to other ovine species and can be used in the clinical investigation of ovine abdominal disorders.     

Evaluation of 3 Assays for Failure of Passive Transfer in Calves

This study examined the sensitivity, specificity, predictive values, and classification accuracy of 3 commonly used screening tests for failure of passive transfer: the sodium sulfite turbidity test, the zinc sulfate turbidity test, and re-fractometry relative to serum immunoglobulin G₁, (IgG₁) concentrations determined by radial immunodiffusion. Serum samples were obtained from 242 calves ranging from 1 to 8 days of age. Using a serum concentration of 1,000 mg/dL IgG₁ to define adequate passive transfer, the zinc sulfate test had a sensitivity of 1.00 and a specificity of 0.52 in the detection of inadequate passive transfer. The endpoint of the test appeared to be higher than desired; calves testing negative had mean serum IgG₁ concentration of 955 mg/dL and a large proportion of calves with adequate passive transfer were misclassified as positive for failure of passive transfer. Using the qualitative zinc sulfate test, the percentage of calves correctly classified with regard to passive transfer status was less than that observed with either the sodium sulfite test or refractometry. The sensitivity of the sodium sulfite assay was 0.85 at a 1+ endpoint and 1.00 at a 2 or 3+ endpoint. The specificity of the sodium sulfite assay varied from 0.87 at a 1+ endpoint and 0.56 at a 2+ endpoint. The sensitivity and specificity of refractometry varied from 0.01 to 1.00 depending on the choice of endpoint. Refractometry correctly classified the largest proportion of calves with regard to their passive transfer status at test endpoints of 5.0 and 5.5 g/dL, 83% and 82% respectively. The highest percentages of calves correctly classified occurred with the sodium sulfite test using a 1+ endpoint (86.30%) and refractometry using a 5.0 g/dL endpoint (83.00%). A regression equation was developed that permitted calculation of an optimal endpoint for refractometric determinations of total serum protein concentration. A serum protein concentration of 5.2 g/dL was equivalent to 1,000 mg/dL serum IgG₁. Optimal selection of tests for passive transfer status in calves will be governed by the prevalence of failure of passive transfer, test performance, and the anticipated costs of classification errors.     

Cytologic analysis of synovial fluid in clinically normal tarsal joints of young camels (Camelus dromedarius)

BACKGROUND: Camels are important in the racing industry and for milk, meat, and hair production in the Middle East. Evaluation of synovial fluid is an important part of the assessment of musculoskeletal injuries in this species. Information in the literature regarding synovial fluid in camels is limited. OBJECTIVES: The objective of this study was to determine the protein and cellular composition of synovial fluid from the tarsal joints of clinically normal, young camels (Camelus dromedarius). METHODS: Thirty clinically healthy, male camels, aged 9 to 12 months, were used in the study. Synovial fluid samples were collected from the right and left tarsal joints. Samples were processed within 60 minutes after collection. Total nucleated cell counts (TNCC) were assessed using a hemacytometer. Total protein concentration was determined using a refractometer. RESULTS: Forty-six samples were analyzed. The TNCC (mean +/- SD) was 175.8 +/- 136.7 cells/microL (range 50-678 cells/microL). Differential cell percentages were obtained for lymphocytes (58.2 +/- 21.55%, range 15-90%), monocyte/macrophages (38.3 +/- 20.8%, range 10-85%), and neutrophils (3.5 +/- 5.1%, range 0-15%). Protein concentration was 2.1 +/- 0.6 g/dL (range 1-3 g/dL). Significant differences were not observed in any parameters between right and left tarsal joints. CONCLUSION: Synovial fluid reference values were established and may be useful in the clinical investigation of joint disease in young camels.     

Effects of time, initial composition, and stabilizing agents on the results of canine cerebrospinal fluid analysis

BACKGROUND: Cerebrospinal fluid (CSF) is considered highly labile, but not all samples are analyzed immediately. Changes in the composition of CSF could potentially affect diagnostic test results and thus influence decisions about patient management. There has been little scientific inquiry into how variables such as time, initial composition, and storage conditions affect results of standard laboratory analysis of CSF. OBJECTIVES: The objectives of this study were to determine the effects of time, protein concentration, and presence or absence of exogenous stabilizing agents on standard CSF analysis results. METHODS: Thirty abnormal CSF samples from 26 dogs were evaluated. Samples were divided into aliquots comprising different treatment groups and stored at 4 degrees C. Total nucleated cell count (TNCC), differential cell count (DCC), and cell morphology were evaluated for all groups; protein concentration was measured for selected groups. Unaltered aliquots were analyzed immediately (T0Hr) and at 2, 4, 8, 12, 24, and 48 hours (T2Hr-T48Hr); aliquots with added fetal calf serum (FCS) or hydroxyethyl starch (hetastarch) were analyzed at T48Hr. RESULTS: Significant time-dependent changes were observed in DCC in unaltered samples. Mononuclear cells deteriorated more rapidly than did neutrophils. Based on microscopic examination and subjective scoring of cell morphology, cells were consistently more degenerate by T24Hr compared with T0Hr. Samples with protein concentrations > or =50 mg/dL were less susceptible to cell deterioration than those with lower protein concentrations. Adding either FCS or hetastarch improved sample stability. CONCLUSIONS: Delayed analysis of canine CSF by 4-8 hours is unlikely to alter diagnostic interpretation, especially for samples with protein concentrations > or =50 mg/dL. The likelihood of misinterpretation is higher for samples with low cellularity or low protein concentration. We provide specific recommendations for adding FCS or hetastarch to samples that will not be analyzed within 1 hour.     

Proteomic analysis of amniotic and allantoic fluid from buffaloes during foetal development

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 μg) was used for in‐solution tryptic digestion, and mass spectrometry analysis (nano‐LC‐MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty‐one proteins were found in the amniotic fluid, and twenty‐one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha‐1‐antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha‐2‐macroglobulin and alpha‐2‐HS‐glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha‐2‐macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.     

卵泡液中总蛋白含量与卵泡直径的关系

通过检测不同直径卵泡的卵泡液中总蛋白含量的变化,从而进一步阐明卵泡液中蛋白成分在卵泡发育过程中的作用。将直径为3~5、5~8、8~10mm的卵泡和囊肿卵泡(>21mm)分为4组(n=10),分别抽取卵泡液。通过TCA/丙酮方法进行样品处理;根据Bradford法检测原理,利用分光光度计对其进行蛋白定量研究。结果表明:3~5mm为4440μg/mL,5~8mm为976μg/mL,8~10mm为686μg/mL,囊肿卵泡(>21mm)为537.2μg/mL。经SPSS13.0软件分析,3~5mm卵泡液中蛋白平均浓度高于其他组(P<0.01);其他3组间差异均不显著(P>0.05);囊肿卵泡中蛋白含量最低。由此可得出,卵泡液中总蛋白浓度随着卵泡直径的增加而降低,表明卵泡液中的蛋白含量与卵泡发育的成熟度呈负相关。而囊肿卵泡的发生很可能和蛋白含量降低有关,为卵泡囊肿发病机制的研究提供新的理论参考。     

Evaluation of five commercially available assays and measurement of serum total protein concentration via refractometry for the diagnosis of failure of passive transfer of immunity in foals

OBJECTIVE: To determine and compare sensitivity, specificity, accuracy, and predictive values of measurement of serum total protein concentration by refractometry as well as 5 commercially available kits for the diagnosis of failure of passive transfer (FPT) of immunity in foals. DESIGN: Prospective study. ANIMALS: 65 foals with various medical problems and 35 clinically normal foals. PROCEDURE: IgG concentration in serum was assessed by use of zinc sulfate turbidity (assay C), glutaraldehyde coagulation (assay D), 2 semiquantitative immunoassays (assays F and G), and a quantitative immunoassay (assay H). Serum total protein concentration was assessed by refractometry. Radial immunodiffusion (assays A and B) was used as the reference method. RESULTS: For detection of IgG < 400 mg/dL, sensitivity of assay H (100%) was not significantly different from that of assays C, E, and G (88.9%). Specificity of assays H (96.0%) and G (95.8%) was significantly higher than that of assays C (79.4%) and E (78.1 %). For detection of IgG < 800 mg/dL, sensitivities of assays H (976%), D (92.9%), C (81.0%), and G (81.0%) were significantly higher than that of assay F (52.4%). Specificity of assays F (100%), G (94.7%), and H (82.8%) was significantly higher than that of assays C (56.9%) and D (58.6%). Serum total protein concentration < or = 4.5 g/dL was suggestive of FPT, whereas values > or = 6.0 g/dL indicated adequate IgG concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Most assays were adequate as initial screening tests. However, their use as a definitive test would result in unnecessary treatment of foals with adequate IgG concentrations.     

Effects of Blood Contamination on Peritoneal D-Dimer Concentration in Horses with Colic

Background: Peritoneal D-Dimer concentration can be determined to assess peritoneal fibrinolysis activity in horses with gastrointestinal disorders. However, blood contamination of peritoneal fluid may occur during collection and could alter peritoneal D-Dimer concentration.
Hypothesis/Objectives: Blood contamination in peritoneal fluid does not affect interpretation of peritoneal D-Dimer concentration in horses with colic.
Animals: Thirty-four horses with colic and 4 healthy horses.
Methods: Peritoneal fluid and blood samples were simultaneously collected upon admission. Then, peritoneal fluid was serially contaminated with the horse's own blood; final contaminations corresponded to 1, 5, 10, and 20% of blood in peritoneal fluid. D-Dimer concentration was determined in blood, peritoneal fluid, and contaminated peritoneal fluid samples. Data were analyzed using a longitudinal linear model and a generalized estimating equations analysis to assess the quantitative and qualitative variations of the effect of blood contamination on peritoneal D-Dimer concentration.
Results: Peritoneal D-Dimer concentration was only quantitatively affected when peritoneal fluid was contaminated at 20% of blood. However, when using increasing cut-off values of peritoneal D-Dimer concentration (100, 2,000, 8,000, and 16,000 ng/mL), this effect disappeared at the highest cut-off values (8,000 and 16,000 ng/mL). When peritoneal fluid contamination was grouped as "minimally contaminated" (≤1% of blood) and "highly contaminated" (≥5% of blood), no significant differences on D-Dimer concentration between both groups at each cut-off value were observed.
Conclusions and Clinical Importance: Although quantitative results of peritoneal D-Dimer concentration could be affected by high levels of blood contamination (≥20%), interpretation of increased peritoneal fibrinolytic activity was not significantly affected.     

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

Concentrations of ketone body and antidiuretic hormone in cerebrospinal fluid in response to the intra‐ruminal administration of butyrate in suckling calves

The aim of the present study was to elucidate the mechanism by which ketone bodies increase antidiuretic hormone (ADH) secretion. Four male Holstein calves (5 weeks of age) were utilized. Four levels of butyrate (0 g, 11 g, 22 g and 44 g) were administrated intra‐ruminally in a 4 × 4 Latin square design and cerebrospinal fluid (CSF, six‐position lumbar puncture), blood plasma and urine were collected. The concentration of total plasma and CSF protein was 5.5–5.6 g/dL and 27.5–28.3 mg/dL, respectively. CSF concentrations of a specific ketone body, 3‐hydroxybutyric acid, were significantly higher in the 22 g and 44 g butyrate groups than in the control group. CSF concentrations of ADH in the 11 g and 44 g butyrate groups were significantly higher than in the control group. Plasma concentration of 3‐hydroxybutyric acid was increased by intraruminal administration of butyrate within 15 min in a dose‐dependent manner, and it was higher in the 22 g and 44 g butyrate group than in the control group from 15 min to 4 h. With the exception of the 11 g butyrate group, plasma concentrations of ADH also increased in response to butyrate treatment, and it was higher in the 44 g butyrate group than in the 22 g butyrate group from 15 min to 1.5 h. The duration of the elevated plasma concentrations of ADH was shorter than that of the plasma concentration of 3‐hydroxybutyric acid. The relationship between the plasma concentrations of ADH and 3‐hydroxybutyric acid was statistically significant but the correlation between the two concentrations was not high. Butyrate treatment elevated the plasma concentration of ADH and also resulted in reduced urine volume and increased urine osmolality. Haematocrit (Ht) values, and the osmolality of CSF and plasma were not different among the groups. Our results suggested that the increased ADH secretion observed in suckling calves fed dry feeds was caused by butyrate‐derived ketone body that crossed the blood‐brain barrier rapidly.