Progressively motile human spermatozoa are well protected against in vitro lipid peroxidation imposed by induced oxidative stress.

Semen samples of 24 patients were analysed. Volumes were measured and the numbers of progressively motile (PMS), motile (MS) and nonmotile spermatozoa (NMS) were determined. These 24 samples appeared to show a large variation in motility percentages and numbers. Spermatozoa of these semen samples were isolated from the seminal plasma and exposed to induced radical oxygen stress imposed by iron/ascorbate. Lipid peroxidation (LPO) was quantified as thiobarbituric acid reactive material. The contributions of PMS, MS and NMS were also estimated. It was found that the PMS did not contribute to the formation of lipid peroxides. The cellular radical defence system of PMS may offer them adequate protection against the harsh conditions of radical oxygen stress. Stepwise regression analyses showed that only the population of NMS contributed significantly to the explanation of the variance in LPO production (R2 = 0.56, P < 0.001). Pre-existing membrane lipid peroxides were not detected in spermatozoa. It is therefore suggested that LPO takes place only after radical oxygen stress has exhausted the cellular defence system. LPO is not the initial, but one of the later, events leading to the death of spermatozoa. It is concluded that the population of progressively motile spermatozoa in semen samples does not contribute to the production of thiobarbituric acid reactive substances as induced by in vitro radical oxygen stress.

[Evaluation of a test strip for the determination of urine specific gravity in the dog]. / Evaluatie van een teststrook voor de bepaling van het soortelijk gewicht van urine van de hond.

The specific gravity of 80 randomly chosen urine samples from healthy dogs without polyuria/polydipsia and from a few dogs with polyuria/polydipsia was measured by refractometry and by test strip method (sg test strip). Urine osmolality and pH were also measured and the urine samples were examined for the presence of glucose. In accordance with earlier observations, there was an excellent correlation between the specific gravity determined by refractory and the osmolality (r = 0.98). The results obtained with the sg test strip correlated poorly with those obtained by refractometry (r = 0.39) and poorly with the osmolality (r = 0.36). In 77.5% of the samples the specific gravity determined with the sg test strip differed by more than 0.005 from that determined by refractometry. After correction of the sg test strip results by +0.005 for urine pH > or = 6.5, this percentage decreased to 60%. The results for the refractometer and sg test strip were significantly different (P < 0.001). These results indicate that the sg test strip investigated is unsuitable for use to determination of the specific gravity of canine urine.

Normal cations and abnormal membrane lipids in the red blood cells of dogs with familial stomatocytosis-hypertrophic gastritis.

Examination of the red blood cells (RBCs) of eight dogs with familial stomatocytosis-hypertrophic gastritis (FS-HG), a multiorgan disease associated with hemolytic anemia, hereditary stomatocytosis (HSt), and hypertrophic gastritis resembling Ménétrier's disease in man, showed abnormal osmotic fragility, normal mean corpuscular volume, slightly increased cell water, and normal cation content and cation fluxes. Cholesterol was decreased in RBC and increased in plasma. In both RBCs and plasma, total phospholipid (PL) was normal, phosphatidylcholine (PC) decreased, and sphingomyelin increased. The palmitic acid content of PC was increased, and the stearic acid content of PC was decreased. Sodium dodecyl sulfate electrophoresis of RBC membrane proteins was normal. These findings have not been described previously in HSt. They suggest that in FS-HG, abnormal composition of the PL in RBCs secondary to abnormal PL in plasma causes defective membrane function and stomatocytic shape-change. This conclusion was supported by a shortened half-life of 51Cr-labeled RBCs from normal dogs after transfusion in dogs with FS-HG. It was concluded (1) that not all hereditary forms of stomatocytosis are necessarily associated with an intrinsic structural defect of the RBC membrane, but that the change in shape of RBC may also be induced by abnormal composition of the plasma; (2) that stomatocytosis may be caused by loss of membrane surface area rather than by the increased cation uptake such as has been shown in some human kindreds with HSt, (3) that FS-HG is a disorder of lipid metabolism, and by consequence, (4) that abnormal lipid metabolism might be involved in the pathogenesis of Ménétrier's disease.

Comparison of different methods of measuring protein and albumin in pigeon sera.

Columbine serum total protein (TP) and albumin concentrations were determined using the biuret method and the bromocresol green dye binding (BCG) method or serum protein electrophoresis on cellulose acetate membranes (SPE). Results obtained using human and pigeon standards were compared. When pigeon albumin was used as a standard. TP values were consistently higher compared with values obtained using human protein as a standard. However, there was a high correlation between the results obtained with the two standards. The correlation between the BCG method and SPE for serum albumin determination was poor, irrespective of the standard used. The method cannot be recommended for pigeon blood. For avian clinical practice it is advised to establish TP concentration using the biuret method and a human standard and to calculate albumin concentration from the results of TP and SPE.

Corticosteroid-induced alkaline phosphatase isoenzyme in the diagnosis of canine hypercorticism.

Corticosteroid-induced isoenzyme of alkaline phosphatase (AP) can easily be demonstrated in canine plasma as a routine procedure because of its greater heat stability at 65 degrees C in comparison with that of other AP-isoenzymes. In this study the accuracy of this test for the diagnosis of hypercorticism was investigated. The AP-65 degrees C test had its highest efficiency when applied to plasma AP levels exceeding 150 units/litre. In a group of 146 dogs, clinically suspected of having hyperadrenocorticism, the test had a sensitivity of 0.92 and a positive predictive value for a positive test result of 0.89. Its lack of specificity (0.44) makes it unsuitable as a diagnostic test. The main application of AP-65 degrees C is in detecting hypercorticism in dogs by routine laboratory measurements, as was demonstrated in 711 dogs, in which a positive predictive value for the presence of hypercorticism of 0.89 was found.

The significance of bile pigment deconjugation by beta-glucuronidase in canine hyperbilirubinemia.

In dogs, the differentiation between haemolytic and cholestatic hepatobiliary diseases cannot be achieved by measuring of the unconjugated:conjugated bilirubin ratio, which is in contrast with generally held clinical concepts. The overlap of the bilirubin ratios between the two groups of icterus-generating diseases might in part be explained by deconjugation of conjugated bilirubin. Enzymatic cleavage by hepatic beta-glucuronidase might result in higher unconjugated bilirubin (UCB) fractions in cholestatic disease. The influence of deconjugation of bilirubins by beta-glucuronidase was investigated in 25 healthy dogs and 35 dogs with spontaneous hyperbilirubinemia due to either hepatobiliary or haemolytic disease. UCB and its mono- and diconjugates were measured by alkaline methanolysis and HPLC in plasma and liver tissue. The activity of beta-glucuronidase was also measured in both liver and plasma. In addition, semiquantitative histochemical quantitation of bilirubins in liver tissue was performed. The concentration and the fraction of UCB in plasma of dogs with hepatobiliary disease were not significantly different from those of dogs with autoimmune haemolytic anaemia. There was a correlation between the fraction of UCB in liver and plasma of jaundiced dogs (r = 0.42, P less than 0.01) and between the histochemically estimated and the biochemically measured total bilirubin concentration in liver tissue. There was no correlation between the beta-glucuronidase activity and either unconjugated or monoconjugated bilirubin in plasma or liver of diseased animals. The fraction and the concentration of UCB in the liver of dogs with hepatic and with haemolytic disease were identical. It is concluded that beta-glucuronidase activity is not the significant factor in explaining the similar levels and fractions of UCB in dogs with hyperbilirubinemia due to either hepatobiliary or haemolytic disease.

[Evaluation of blood chemistry using 'dry chemistry' in small animal practice]. / Evaluatie van bloedonderzoek met behulp van 'droge chemie' in de kleine huisdierenpraktijk.

In this paper three different systems for clinical chemistry determinations by the general practitioner are evaluated. The systems, Seralyzer, Kodak Ektachem DT 60 and Reflotron, are based on the use of dry reagent strips in combination with a reflectometer. The principle of reflectometry is discussed briefly. These systems enable the practitioner to carry out the majority of the common chemical laboratory blood tests quickly and with an acceptable degree of confidence. The present possibilities are given for each system, including the costs of instruments and tests in the Netherlands. The results of some common tests in small animal medicine have been compared with standard methods in our own laboratory. Since in a few tests species dependent differences were found, it is recommended that each dry chemistry test should be carefully evaluated for each animal species.

Collection and analysis of urine from racing pigeons (Columba livia domestica).

Urine from racing pigeons (Columba livia domestica) was collected by a method using cloacal cannulation. Reference values for 12 chemical and physical variables in urine of racing pigeons were determined for use in clinical practice.

A water deprivation test for the differentiation of polyuric disorders in birds.

A water deprivation test was developed for the differentiation of polyuric disorders in birds using the racing pigeon as a model. For a period of 3 days of food and water deprivation, urine and plasma osmolalities and body weight of 40 clinically healthy racing pigeons were monitored. Reference values for urine osmolality after 40 hours of food deprivation were 79 to 480 mOsmol/kg. After 64 hours of food deprivation and 24 hours of water deprivation these values were 450 to 875 mOsmol/kg. No significant rise in urine osmolality was seen after the first 24 hours of water deprivation. It is concluded that an urine osmolality greater than 450 mOsmol/kg is indicative of a normal concentrating capacity of the kidney in the pigeon. Data from the literature suggest that these values can be applied to other avian species.

Enzyme activities in tissues and elimination half-lives of homologous muscle and liver enzymes in the racing pigeon (Columba Livia domestica).

Tissue enzyme profiles of heart, liver, pectoral muscle, quadriceps muscle, duodenum, kidney and brain from racing pigeons were established. The enzymes were alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), gamma glutamyltransferase (y-GT), alkaline phosphatase (AP), and creatine kinase (CK). Elimination half-lives (tybeta) of certain enzymes were also determined. The mean values (+/- SD) were: ASAT, 7.66 +/- 1.55 (liver) and 6.51 +/- 0.83 (muscle); ALAT, 15.69 + 1.70 (liver) and 11.99 +/- 1.32 (muscle); LDH, 0.71 + 0.10 (liver) and 0.48 +/- 0.07 (muscle); GLDH, 0.68 +/- 0.17 (liver), CK 3.07 +/- 0.59 hours (muscle). GLDH is the most liver-specific enzyme in the pigeon, but increased activities in the plasma are likely only in the acute stage of severe liver cell damage, since this enzyme is localised within the mitochondria and has a short half life. LDH and ASAT seem to be the most sensitive indicators of liver cell damage, though contributions come from muscle damage. Muscle cell damage can be differentiated from liver cell damage by measuring plasma CK activity, since CK is both a specific and a sensitive indicator of muscle cell damage. In a clinical setting the combined use of LDH, ASAT and CK permits differentiation between liver and muscle cell damage in racing pigeons.

Esterases in inbred strains of mice with differential cholesterolemic responses to a high-cholesterol diet.

Specific esterase isoenzyme patterns in plasma may be associated with responsiveness of serum cholesterol to dietary cholesterol. In rabbits and rats the presence and absence of a high-mobility, anodal esterase band on electrophoresis have been shown to be associated with hypo- and hyperresponsiveness, respectively. We fed for 28 days male mice of 7 inbred strains either a low-cholesterol, commercial diet or a diet containing 2% (w/w) cholesterol, 0.5% cholic acid and 5% olive oil. Feeding the high-cholesterol diet revealed marked inter-strain differences in the responses of plasma and liver cholesterol; the increases ranged from 21 to 129% and from 10 to 80-fold, respectively. There was no association between esterase isoenzyme patterns in plasma and the sensitivity to the high-cholesterol diet. The mean baseline plasma total esterase activity tended to be positively associated with the absolute response of plasma cholesterol to the high-cholesterol diet (r = 0.56; n = 7), but the positive relationship between the baseline concentration of the ES-1 component in plasma and the cholesterolemic response was stronger (r = 0.84; n = 7; P less than 0.05). The high-cholesterol diet caused a significant increase in plasma total esterase activities in 6 out of the 7 strains. Evidence is presented that the increase in plasma total esterase activity, which was associated with an increase in the activity and concentration of the so-called ES-2 isoenzyme, is the result of an enhanced release of esterases from the intestine, rather than from the liver. A significant, positive correlation was found between the baseline intestinal esterase activity and the cholesterolemic response after cholesterol feeding (r = 0.83; n = 7; P less than 0.05).

Dietary cholesterol-fat type combinations and carbohydrate and lipid metabolism in rats and mice.

In rats fed semipurified cholesterol-free diets, dietary corn oil induced higher levels of liver cholesterol, but lower concentrations of plasma cholesterol, plasma triglycerides and blood D-3-hydroxybutyric acid, than did coconut fat. Addition of cholesterol to the diets (1%, w/w) greatly increased liver cholesterol levels but did not affect the corn oil effect upon liver and plasma cholesterol. In contrast, dietary cholesterol prevented the corn oil effects upon plasma triglycerides and blood D-3-hydroxybutyric acid. Liver glycogen levels in rats increased significantly after cholesterol feeding, the effect being independent of dietary fat type. In mice, dietary corn oil, compared to coconut fat, elevated liver cholesterol only when the diet contained cholesterol. Corn oil caused an increase of liver glycogen in mice in the presence, but not in the absence of dietary cholesterol. This study shows that dietary cholesterol-fat type combinations influence various aspects of lipid and carbohydrate metabolism in rats and mice.

Cholesterol metabolism and esterases in four strains of rats with differential cholesterolemic responses to a high-cholesterol, high-cholate diet.

The increase in serum cholesterol after feeding a diet containing 2% (w/w) of cholesterol and 0.5% of cholate for 13 days was 200 and 800% in two hypo- and two hyper-responsive inbred strains of rats, respectively. While remaining on the high-cholesterol, high-cholate diet for longer periods, the level of serum cholesterol dropped in the hyper-responsive strains, and after 8 weeks on the diet one hyper-responsive strain had similar serum cholesterol concentrations as the two hypo-responsive strains. The feeding of a semipurified diet, containing 1% (w/w) of cholesterol and 20% of fat, did not discriminate between the two hypo- and hyper-responsive strains with respect to the response of serum cholesterol. The activities in plasma of the indicators for liver function, aspartate amino transferase and alkaline phosphatase, were significantly increased in all strains after feeding the high-cholesterol, high-cholate diet. Only alkaline phosphatase was increased by the semipurified diet. Evidence is presented that in the four inbred strains of rats the differential cholesterolemic response to the high-cholesterol, high-cholate diet is not related to the baseline serum lipoprotein profile, liver cholesterol accumulation, fecal bile acid excretion, and the total activities and patterns of esterases in serum, liver and small intestine.

Separation and heat stability of the corticosteroid-induced and hepatic alkaline phosphatase isoenzymes in canine plasma.

A convenient method has been developed for the separation of alkaline phosphates (AP) isoenzymes from canine plasma. The various forms of AP activity were extracted by ethanol and separated on an anion exchanger by fast protein liquid chromatography. In this way a complete discrimination was achieved between the increase in plasma AP activity due to liver disease and that due to corticosteroid induction. The corticosteroid-induced form of AP could be separated from the other isoenzymes because of its relative heat stability at 65 degrees C. A quantitation of the contribution of liver and corticosteroid-induced AP isoenzymes to the total plasma AP activity could be made from the respective heat inactivation plots. The separation of the isoenzymes may be valuable in the purification of the different isoenzymes for further characterization.

The effect of long-term fasting on ketone body metabolism in the dog.

Ketone-body metabolism was studied in overnight-fasted and in 10-days fasted dogs by a ketone-body tolerance test and by infusing [14C]ketone bodies. Clearance of ketone bodies from the blood was significantly decreased after 10 days of fasting. The utilization of ketone bodies was, however, significantly higher in the fasted animals due to the increase in blood ketone-body concentrations. It is concluded that the low level of ketone bodies, which is characteristic for fasting dogs, results from an efficient peripheral utilization. The contribution of ketone bodies to the daily energy requirement of the dog has been tentatively estimated to increase from 7% in the overnight-fasted state to 13% after 10 days of starvation.

Treatment of young rats with cholestyramine or a hypercholesterolemic diet does not influence the response of serum cholesterol to dietary cholesterol in later life.

Groups of 10 female Wistar rats (aged 4 weeks) were fed for 29 days either a low-cholesterol commercial diet, a commercial diet containing 2% (w/w) cholesterol, 0.5% cholate and 5% olive oil or a diet containing 2% cholestyramine. The rats were then fed the low-cholesterol commercial diet for the next 91 days and the high-cholesterol diet for another 29 days. There was no significant difference between the groups in the increase of cholesterol in serum and liver during the last period of cholesterol feeding. A fourth group of 10 animals was fed the diet containing cholesterol and cholate during the entire experimental period of 149 days. By the end of the experiment serum cholesterol in these animals was lower and liver cholesterol was higher than in the 3 groups fed the high-cholesterol diet during days 120-149 of the experiment. This study does not present evidence for imprinting effects of early diet manipulation on the later cholesterolemic response to a high cholesterol diet.

Blood chemistry reference values in racing pigeons (Columba livia domestica).

Reference values for 23 blood chemical parameters in racing pigeons (Columba livia domestica) were established for use in clinical pathology. The inner limits are given for the percentiles P(2).(5) and P(97.5) with a probability of 90%. A minimum of 50 blood samples collected between September and January from different animals aged between 6 months and 12 years (median 1.5 years) was used for each parameter. Reference values obtained in the present study are compared with values published previously.

Evaluation of the refractometric method for the determination of total protein in avian plasma or serum.

Serum total protein concentrations in pigeon blood determined with the biuret method (TPB-se) were compared with total protein concentrations in plasma (TPR-pl) and serum (TPR-se) obtained by estimation from refractive index. The refractometric method consistently yielded higher values (P<0.01). A significant correlation between TPB-se and TPR-se was found (r = 0.62) but not between TPB-se and TPR-pl. The calculated regression equation TPR-se = 0.69 TPB-se + 16 was considered to have no practical value, because the correlation coefficient was low (r = 0.62). The refractometric method for determination of TP in pigeon blood is not recommended.

Insulin-like growth factor I and growth hormone in canine starvation.

The roles of plasma insulin-like growth factor I (IGF I) and growth hormone (GH) were studied in 7 beagle dogs before and during starvation and during refeeding. IGF I levels significantly decreased from 75.2 +/- 5.9 ng/ml at 7 days prior to the start of starvation to 9 +/- 1.7 ng/ml at 19 days after the commencement of starvation (mean +/- SEM; P less than 0.0001). During refeeding IGF I significantly rose from 9 +/- 1.7 ng/ml to 55.5 +/- 7.5 ng/ml within 9 days (mean +/- SEM; P less than 0.002). During starvation plasma GH levels significantly increased (P less than 0.05) and these elevated levels returned to normal during refeeding. The dogs' GH secretory capacity significantly increased during starvation (P = 0.012) and became normal again during refeeding. The following conclusions can be drawn from this study: 1) starvation in the dog leads to a significant and drastic reduction of the circulating levels of IGF I, and 2) starvation in the dog, as in man, leads to increased circulating GH levels and to an increased GH-secretory capacity possibly brought about by a lack of a negative feedback normally exerted by IGF I.