Infectious stunting and leg weakness in broilers: I. Pathology and biochemical changes in blood plasma

A syndrome of stunting and leg weakness could be reproduced experimentally by inoculation of 1-day-old broilers with homogenised intestines from affected birds. Inoculated birds kept in isolators showed highly impaired growth until 3 weeks p.i. Birds produced mucoid yellowish coloured droppings and at post mortem thin liquid intestinal contents were found. Biochemical examination of blood plasma showed low plasma carotenoid concentrations and an increased alkaline phosphatase (ALP) activity, mainly caused by one isoenzyme, which was most likely of intestinal origin. These findings implicate infectious causal agents with the intestines as the site of primary involvement. Bone abnormalities consisted of rickets-like changes at the age of 3 weeks, whereas a distinct dyschondroplasia was seen at 4 weeks. The syndrome could also be transmitted to uninoculated birds kept in contact with birds inoculated at 1 day of age. Birds inoculated at 7 days of age also showed greatly impaired growth but developed no macroscopical bone disorders. Inoculation at 14 days of age did not result in impaired growth or bone abnormalities. Following inoculation with REO virus, isolated from a field case, no bone abnormalities occurred. However, a shortlived impaired growth, diarrhoea, increased plasma ALP activity and decreased carotenoid concentration were observed. The rapid spread of the disease and the role of REO virus are discussed.     

Plasma alkaline phosphatase isoenzymes in hepatobiliary disease

BY CELLULOSE ACETATE OR ACRYLAMIDE GEL ELECTROPHORESIS IT IS POSSIBLE TO SEPARATE THESE ALKALINE PHOSPHATASE ISOENZYMES FROM SERUM: [anode] fast liver, slow liver, placenta/Regan, bone, intestine, bile [cathode]. Heat or chemical inhibition can confirm the differentiation.Normal adult serum always contains slow-liver isoenzyme, and sometimes bone isoenzyme: the latter is always present in serum of children. In hepatobiliary disease slow-liver isoenzyme was always increased: intestinal isoenzyme appeared in many cases of cirrhosis (of blood groups B and 0) but fast-liver and bile isoenzymes were occasionally seen in miscellaneous cases. The findings in other diseases included Regan isoenzyme in six out of 45 cases of malignant disease.     

Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made.     

Plasma intestinal alkaline phosphatase isoenzymes in neonates with bowel necrosis.

AIM--To determine if the intestinal isoenzymes of alkaline phosphatase (ALP) are biochemical markers of bowel necrosis in neonates. METHODS--Plasma ALP isoenzymes were measured in 22 babies with bowel necrosis, histologically confirmed, and in 22 matched controls. The isoenzymes were also measured in 16 infants with signs of necrotising enterocolitis, who recovered without histological confirmation of bowel necrosis. The isoenzymes were separated by polyacrylamide gel electrophoresis. Auxiliary tests for identification included neuraminidase digestion and treatment with monoclonal and polyclonal antiplacental antibodies. RESULTS--Intestinal ALP was detected in 16 infants with bowel necrosis--13 had fetal intestinal ALP (FI-ALP) and three had adult intestinal ALP (AI-ALP). FI-ALP was detected in nine of the controls. In the babies with bowel necrosis intestinal ALP was found over all gestations, but in the controls only in those less than 34 weeks. The percentages of total ALP activity due to intestinal ALP were significantly higher in those with bowel necrosis compared with matched controls (p = 0.028). In babies of all gestations diagnostic sensitivity for the presence of intestinal ALP as a marker of bowel necrosis was 73% and diagnostic specificity 59%. In babies greater than 34 weeks' gestation, diagnostic sensitivity fell to 60% but the test became completely specific. In two babies FI-ALP increased from zero/trace to high activity coincident with the episode of bowel necrosis. In 16 babies with signs of necrotising enterocolitis but unconfirmed bowel necrosis FI-ALP was detected in four. CONCLUSION--Intestinal ALP seems to be released into the circulation in some babies with bowel necrosis, but its detection does not have the diagnostic sensitivity and specificity to be a reliable biochemical marker of the condition.     

Chemical inhibition method for alkaline phosphatase isoenzymes in human serum.

The authors adapted a chemical inhibition procedure using L-phenylalanine and urea as specific inhibitors to quantitate the activities of bone, liver, and intestinal alkaline phosphatase (ALP) isoenzymes in human serum. The results of this assay were compared with electrophoretic separation, gamma-glutamyl transpeptidase (GGTP) activity, and the clinical setting in a group of patients with elevated total ALP activity. In addition, expected ranges of serum ALP isoenzymes for healthy young men and also for a geriatric population are presented.     

Polyacrylamide gel disc electrophoresis of alkaline phosphatase isoenzymes in bone and liver disease.

Acrylamide gel disc electrophoresis provides a reliable and reasonably rapid method of differentiating the raised serum alkaline phosphatase (AP) of bone origin from that of liver origin. The technique has been placed for the first time on a semiquantitative basis. Measurement of both band width and band position effectively distinguishes the bone from the liver isoenzyme, but band width provides superior discrimination. An origin band was seen in none of the normal subjects and in only 7% of patients with bone disease but was present in 78% of patients with liver disease, a highly significant increase. Fifty percent of normal individuals had a small-intestinal band in serum taken two hours after a meal, as did 35% of patients with liver disease, but the incidence of intestinal bands in bone disease was only 11%, significantly less than in the other two groups. The genetic control of small-intestinal AP in serum has been confirmed, but it has been demonstrated that the decrease of intestinal AP in bone disorders is not genetically determined.     

Evaluation of two new methods for routine measurement of alkaline phosphatase isoenzymes.

AIMS: To evaluate the performance of two new methods for the analysis of alkaline phosphatase (ALP) isoenzymes designed for use in the routine chemical pathology laboratory: pre-incubation with neuraminidase before agarose electrophoresis; and selective precipitation of the bone isoenzyme with wheat germ agglutinin (WGA). METHODS: Serum samples from 39 patients were analysed. Seventeen were from patients with liver disease, eight from patients with bone disease, and 14 from patients with normal ALP activity and no evidence of liver or bone disease. The two new methods were compared with the established method, wheat germ agglutinin affinity electrophoresis. RESULTS: There was good correlation between the neuraminidase and WGA electrophoretic methods. The WGA precipitation method showed negative interference in the measurement of bone isoenzyme activity in samples containing biliary alkaline phosphatase. Both the new methods had advantages of speed and simplicity over the existing method, but cost per test was considerably higher. CONCLUSIONS: The neuraminidase electrophoretic method is a satisfactory alternative to the WGA affinity electrophoretic method, although it is more expensive. The WGA precipitation method cannot be recommended for use with serum samples from patients with suspected liver disease.     

Purification and kinetic study of bone and liver alkaline phosphatase isoenzymes in the dog

To study bone and liver alkaline phosphatases (ALP) in the dog, tissue samples were obtained immediately after death from dogs apparently involved in road traffic accidents. Tissue samples were homogenized, and extracted protein was subjected to diethylaminoethyl cellulose–Sephadex column chromatography equilibrated previously with 0.01 M Tris-hydrochloric acid buffer (pH 7.2) and eluted with linear gradient of ionic strength from 0 to 0.4 M NaCl. Fractions with peak activity of ALP were considered a source of purified enzyme. ALP activity was measured by a kinetic method using p-nitrophenylphosphate as a substrate. The kinetic study was performed using a constant concentration of purified isoenzymes and different concentrations of the substrate. Precise Km values for liver and bone ALP were identified using direct linear plot (Eisenthal–Cornish-Bowden plot). Our study showed that the Km value of liver isoenzyme was 13 times greater than that of bone isoenzyme (3.3 mmol and 0.25 mmol, respectively). This may be due to the physiological role of bone ALP in preparing phosphate for bone mineralization and the retention of phosphates in mineral complexes with calcium in bone tissues.     

Influences of uncommon isoenzymes on determination of alkaline phosphatase activity by dry-chemistry analyzers

Dry-chemistry(DC) analysis may be influenced by some matrix effects for measuring uncommon isoenzyme forms. Placental and intestinal alkaline phosphatase(AP) are overestimated by the VITROS DC, compared with results obtained with the wet-chemistry(WC) method of Bretaudiere, et al. using 2-amino-2-methyl-1-propanol (AMP) buffer, however, no such discrepancy between AP results in any DC method and that with a routine WC method recommended by Japanese Society of Clinical Chemistry in that 2-ethylaminoethanol(EAE) buffer is used, has been demonstrated. The type of buffer used affects differently the rates of AP isoenzymes activities. We therefore examined whether the presence of uncommon AP isoenzyme forms in serum caused aberrant DC results for AP in comparison with a routine WC method using EAE buffer. Here, serum samples with only liver AP and bone AP(n : 32); high-molecular-mass AP(n : 11); placental AP(n : 12); intestinal AP(n : 13) and immunoglobulin (Ig) bound AP(n : 12) were analyzed for total AP activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM 4410 and a WC analyzer: HITACHI 7350. Values obtained in all of the DCs for sera containing only liver/bone AP agreed with those with the WC method. For sera containing placental AP, the VITROS values were higher than those with the WC method, while the FUJIDRYCHEM values and the SPOTCHEM values were lower. The VITROS values and the FUJIDRYCHEM values for sera containing intestinal AP were lower than those with the WC method, while the SPOTCHEM values were higher. All of the DCs did not affect high-molecular-mass AP and Ig bound liver/bone AP types of macro AP, but underestimated Ig bound intestinal type. Ig bound intestinal AP may be sieved by DC multilayer elements.     

Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase.

The potential use of the protein denaturant guanidine hydrochloride to inhibit selectively the enzyme activity of serum alkaline phosphatase isoenzymes from liver, bone, intestine, and placenta was investigated. Inhibition of each isoenzyme was shown to be dependent on time and concentration of inhibitor. In the presence of 0.3 mol/l (28.7 g/l) guanidine hydrochloride for 170 seconds 14%, 47%, and 90% of the total alkaline phosphatase activity remained in samples of bone, liver, and intestinal origins, respectively. In contrast, the activity of the placental isoenzyme increased by 24%. The degree of inhibition was shown to be independent of total alkaline phosphatase activity. Investigations were performed at 37 degrees C using the Cobas Bio centrifugal analyser. We conclude that this reagent has several practical advantages over urea as a selective inhibitor of alkaline phosphatase isoenzymes, including a faster and more reproducible inhibition at a much lower reagent concentration.     

Agarose gel patterns of alkaline phosphatase isoenzymes before and after treatment with neuraminidase

The clinical value of alkaline phosphatase isoenzyme analysis is limited by the inability of most electrophoretic methods to resolve the liver and bone isoenzymes. The authors attacked this problem by treating serum samples with neuraminidase, then running treated and untreated samples side-by-side on specially prepared agarose gels. Each isoenzyme showed a characteristic mobility before and after neuraminidase treatment that allowed its identification. The mobility of the bone isoenzyme was most affected, whereas the intestinal isoenzyme was resistant to the action of neuraminidase. In samples with both liver and bone isoenzymes, pretreatment with neuraminidase clearly distinguished the bands, allowing quantitation by densitometry. Using this method, the authors discovered 22 liver isoenzymes in 54 samples that were interpreted as only bone isoenzyme before neuraminidase treatment. They also detected two bone isoenzymes in 35 samples that appeared to contain only liver +/- biliary isoenzymes. In addition, this procedure enabled them to characterize several unusual isoenzymes with respect to mobility, thus avoiding confusion with the other isoenzymes.     

Measurement of alkaline phosphatase of intestinal origin in plasma by p-bromotetramisole inhibition.

L-p-bromotetramisole was used to inhibit non-intestinal alkaline phosphatase (of liver or bone origin) (EC 3.1.3.1; ALP) in plasma, and intestinal ALP was measured from the uninhibited activity. The method of determination is convenient and correlated well with measurement by immunocapture assay. If carried out in parallel with wheat-germ lectin precipitation of bone ALP, subtraction of intestinal ALP activity from that of non-bone ALP in the supernatant can be used to measure the ALP that originates from the liver in men and non-pregnant women.     

Intestinal origin alkaline phosphatase activity in plasma for differential diagnosis of jaundice.

Intestinal origin alkaline phosphatase activity (ALP) in plasma was measured by a sensitive immunocapture assay in 104 jaundiced patients--84 with intrahepatic and 20 with post-hepatic jaundice. Increased enzyme activities were observed in those with intrahepatic disease and subnormal values in those with post-hepatic disease. At a discriminant level intestinal origin ALP showed a diagnostic sensitivity of 77% for intrahepatic cholestasis, with a diagnostic accuracy of 75% for its differentiation from post-hepatic jaundice. This diagnostic accuracy is not as good as that derived with other techniques--for example, imaging--and the technique is therefore not recommended as a supplement or replacement.