A critical examination of the value of combined determinations of lecithin:cholesterol acyltransferase and lipoprotein-X in the differential diagnosis of liver disease.

The suggestion that combined determinations of lecithin: cholesterol acyltransferase activity and lipoprotein-X can be used to distinguish between patients with intrahepatic cholestasis and extrahepatic biliary obstruction has been studied. Of 21 patients who were lipoprotein-X positive and in whom an unequivocal diagnosis was made, 11 had intrahepatic cholestasis and 10 extrahepatic obstruction. The range of plasma lecithin:cholesterol acyltransferase activity was similar in the two groups and did not enable distinction to be made between them.     

The incidence and likely origins of serum particulate alkaline phosphatase and lipoprotein-X in liver disease

Both serum particulate alkaline phosphatase and serum lipoprotein-X have been proposed as diagnostic markers for obstructive liver disease. In this study their diagnostic efficiencies have been compared with other biochemical indicators of liver function and the relative incidence of these two markers has been determined in 241 patients with well defined liver disease. Although these markers appeared together most frequently in obstructive liver disease they were both present in some patients with liver disorders where obstruction was unlikely. One marker was present, independently of the other in 35% of the patients studied, mainly those without apparent cholestasis.The reasons for this are discussed in terms of the likely origins of the two markers and the probability that serum particulate alkaline phosphatase exists in two forms, as part of a multienzyme plasma membrane complex and as soluble liver enzyme associated with lipoprotein-X.     

Study of the abnormal lipoprotein-X in obstructive and non-obstructive jaundice

A lipoprotein-X (LP-X) test has been performed in normal subjects and in patients with hepatobiliary diseases and haemolytic anaemia.Statistical analysis revealed a correlation between the occurrence of LP-X and increase of conjugated bilirubin and alkaline phosphatase.The LP-X test makes it possible to differentiate obstructive from non-obstructive jaundice but it fails to demonstrate or exclude cholestasis in all cases and cannot distinguish between intrahepatic and extrahepatic cholestasis.     

Jaundice.

Jaundice is a disorder of bilirubin metabolism and has many causes. History and physical examination help establish the diagnosis in 70 to 80 percent of patients. Elevation of alkaline phosphatase and gamma-glutamyl transpeptidase suggests cholestasis, either intrahepatic (e.g., medication reactions) or extrahepatic (e.g., choledocholithiasis), whereas markedly elevated serum aminotransferases are indicative of hepatocellular damage from infection, toxins or ischemia. Ultrasound examination is a useful initial procedure when extrahepatic obstruction is suspected. Endoscopic retrograde cholangiopancreatography and computed tomography may be better used to diagnose obstruction at the level of the pancreas or distal common bile duct. The treatment is based on the etiology of jaundice and includes removal of offending medications or toxins, therapy for underlying liver disease or surgery for extrahepatic obstruction.     

Multiple forms of gamma-glutamyltransferase: a clinical study

We have measured the amounts of different molecular forms of gamma-glutamyltransferase (EC 2.3.2.2), leucine aminopeptidase (EC 3.4.11.2), and alkaline phosphatase (EC 3.1.3.1) in serum of patients with different types of liver disease. A high-molecular-mass (greater than 1 000 000 Da) form of gamma-glutamyltransferase and of each of the other enzymes is present in greatest amounts in patients with jaundice from extrahepatic obstruction. A gamma-glutamyltransferase form of intermediate molecular mass (250 000 to 500 000 Da) is present in the serum from most patients with liver disease and can be separated by electrophoresis into several bands. We found that one of these bands predominated in patients with extrahepatic obstructive jaundice, whereas the others predominated in patients with other liver diseases. Electrophoresis of serum gamma-glutamyltransferase may be of clinical value in distinguishing extrahepatic from intrahepatic causes of jaundice.     

Graphical analysis of laboratory data in the differential diagnosis of cholestasis: a computer-assisted prospective study

Data on 15 laboratory analytes obtained in 145 prospectively investigated cholestatic patients with viral hepatitis, chronic intrahepatic cholestasis and extrahepatic biliary obstruction were submitted to a computer-based graphical evaluation using probabilistic test analysis. This revealed a marginal utility for alkaline phosphatase, gamma-glutamyltransferase and the direct/total bilirubin ratio at specific cut-off points for the exclusion of extrahepatic cholestasis (PVneg 90%-100%). Aspartate aminotransferase and alanine aminotransferase values with cut-off points at 200 U/l and 300 U/l, respectively, were powerful discriminators between acute viral hepatitis and the other disease categories, while lactate dehydrogenase, erythrocyte sedimentation rate and the ratios gamma-glutamyltransferase/alanine aminotransferase as well as total bilirubin/gamma-glutamyltransferase were useful at specific cut-off points indicating the absence of this diagnosis (PVneg 92%-100%). An aspartate aminotransferase/alanine aminotransferase ratio above 1.5 and serum gamma-globulin concentrations above 20 g/l strongly suggested cholestasis due to chronic parenchymal liver disease (PVpos 92% and 90%, respectively). This graphical approach to laboratory data analysis enhances the understanding of the interrelations between cut-off points and sensitivity, specificity and predictive values and also of the influence of disease prevalence on disease prediction. It also adds to present knowledge by demonstrating the clinical relevance of several readily available, albeit rarely utilized diagnostic analytes.     

Lipoprotein-X and diagnosis of cholestasis: comparison with other biochemical parameters and liver biopsy.

The presence or absence of histological signs of cholestasis (on the basis of liver specimens obtained by means of liver biopsy) was compared with total bilirubin, alkaline phosphatase, gamma-glutamyl transpeptidase, ornithine carbamoyltransferase, serum glutamic oxaloacetic transaminase levels and LP-X test in 157 patients suffering from different liver diseases. The LP-X test was positive in 93% of the 59 cases in whom histological evidence of cholestasis was observed and negative 95% of the 98 cases in whom histological examination was negative. LP-X concurs more frequently with the histological picture than do total bilirubin and alkaline phosphatase. These data confirm that LP-X test is more specific than the tests traditionally used to demonstrate or exclude cholestasis. An increment in gamma-GT levels was observed in 97% of the patients with a positive LP-X test. These clinical results have been discussed in the light of recent data regarding the mechanism of lipoprotein-X formation and the possible relationships between LP-X and gamma-glutamyl transpeptidase.     

Comparison of alkaline phosphatase isoenzymes determined by an inhibition method and by electrophoresis

A chemical inhibition procedure suitable for the routine determination of alkaline phosphatase (AP) isoenzymes in serum has been adapted for use with a fast kinetic analyzer, System Olli 3000. The results of this procedure are compared with the electrophoretic separation of alkaline phosphatase isoenzymes. The comparison of the results obtained indicates that the AP-urea/AP ratio can be used to differentiate between patients with bone and liver disease and that it is possible to estimate the relative bone and liver isoenzyme activities from this ratio quickly using two simple equations.     

Serum bile acids

Summary Serum bile acid measurements now available by radioimmunoassay have proven to be the most sensitive procedure developed to assess diseases of the hepatobiliary system in both adult and pediatric liver disease. Their clinical utility appears to hold particular promise in SGOT, alkaline phosphatase, bilirubin and albumin are still normal. Serum bile acid determinations have been shown to be particularly useful in the diagnosis of alcoholic liver disease, drug-induced liver disease, viral hepatitis and cholestasis of intra- and extrahepatic origin. In infants, serum bile acid measurements can be used to establish the diagnosis of biliary atresia. When serum bile acids are determined post-prandially, they are the most sensitive indicator of liver dysfunction developed to date.     

Clinical significance of serum glutathione reductase in various clinical conditions, especially in liver diseases.

Serum glutathione reductase activity was measured in various conditions including acute hepatitis, chronic hepatitis, liver cirrhosis, malignant neoplastic diseases, and obstructive jaundice. A statistically significant elevation of the enzyme activity was found in all of these clinical conditions above normal value, especially in patients with acute hepatitis, some liver cancer, and malignant biliary obstruction. Comparison with other liver function tests showed the existence of statistically significant correlations of serum glutathione reductase with SGOT, SGPT and alkaline phosphatase in acute hepatitis, and with alkaline phosphatase in cirrhosis. In parenchymatous liver disease, serial determination was found to be important. High values in obstructive jaundice suggest the malignant obstruction.     

Electrophoresis of gamma-glutamyltranspeptidase on cellogel. The appearance of the alpha2-beta band in positive LP-X sera.

Fractionations of serum gamma-glutamyltranspeptidase (gamma-GT) and determinations of the "abnormal serum lipoprotein X" (LP-X) have been carried out in sera from patients with different hepatobiliary disorders. LP-X was used to demonstrate or exclude cholestasis. One gamma-GT fraction, alpha2-beta, may be of interest to distinguish between extrahepatic obstruction and intrahepatic cholestasis as was revealed by statistical analysis.     

Clinical significance of serum high-molecular-mass alkaline phosphatase,alkaline phosphatase–lipoprotein-X complex,and intestinal variant alkaline phosphatase

This paper is a study to identify the clinical significance of high-molecular-mass alkaline phosphatase (ALP:E:C.3.1.3.1.), ALP–lipoprotein–X complex (LP-X) and intestinal variant ALP. We used cellulose acetate and agarose gels and techniques including wheat germ lectin, cetavlon-diethyl ether, thermostatability, neuraminidase and L -phenylalanine to improve the electrophoretic separation of the alkaline phosphatase isoenzymes. Patients' serum samples were electrophoresed from a diverse group of individuals ill with cholestasis, neoplastic disease metastatic to the liver, hepatocellular carcinoma, cirrhosis, diabetes mellitus, and chronic renal disease. Agarose gels provided better separation of ALP isoenzymes than cellulose acetate gels. The results also indicated that high-molecular mass ALP is present in patient's serum in conditions associated with cholestasis especially caused by hepatic malignancy. High-molecular mass ALP was frequently found to co-exist with the liver isoenzyme and LP–XALP complex. The intestinal variant was identified in patients with malignancy, cirrhosis, chronic renal disease and diabetes mellitus. Intestinal ALP coexisted concomitantly with a variant intestinal ALP. Intestinal variant ALP is most likely composed of intestinal ALP attached to a cellular membrane-binding domain, or may be an artifact produced by neuraminidase incubation © 1994 Wiley-Liss, Inc.     

Rate of in vitro Incorporation of Precursors into Hepatic Lipids and Proteins in Patients with Extrahepatic Cholestasis

Abstract. Extrahepatic cholestasis in man is known to be associated with high phosphoglyceride and cholesterol concentrations in serum. Moreover, an abnormal lipoprotein, often referred to as lipoprotein-X (LP-X), appears in serum in cases of extrahepatic cholestasis.
The purpose of this study was to determine the rate of incorporation of precursors into hepatic lipids and proteins in vitro and to compare these changes with the lipoproteins in plasma in patients with extrahepatic cholestasis. The rate of incorporation of glycerol into triglycerides and into different classes of phosphoglycerides was determined. The rate of incorporation of leucine into proteins was also determined in liver slices from 15 patients with extrahepatic cholestasis and compared with controls. In cholestatic liver tissue, the rate of incorporation of glycerol into choline phosphoglycerides and into ethanolamine phosphoglycerides was significantly increased. The rate of incorporation of leucine into hepatic proteins was also increased significantly in these patients. The results suggest that the changed metabolism of phospholipids and protein in liver tissue in cholestatic conditions may contribute to the changes in the serum lipoprotein pattern.     

Rate of in vitro incorporation of precursors into hepatic lipids and proteins in patients with extrahepatic cholestasis

Abstract. Extrahepatic cholestasis in man is known to be associated with high phosphoglyceride and cholesterol concentrations in serum. Moreover, an abnormal lipoprotein, often referred to as lipoprotein-X (LP-X), appears in serum in cases of extrahepatic cholestasis. The purpose of this study was to determine the rate of incorporation of precursors into hepatic lipids and proteins in vitro and to compare these changes with the lipoproteins in plasma in patients with extrahepatic cholestasis. The rate of incorporation of glycerol into triglycerides and into different classes of phosphoglycerides was determined. The rate of incorporation of leucine into proteins was also determined in liver slices from 15 patients with extrahepatic cholestasis and compared with controls. In cholestatic liver tissue, the rate of incorporation of glycerol into choline phosphoglycerides and into ethanolamine phosphoglycerides was significantly increased. The rate of incorporation of leucine into hepatic proteins was also increased significantly in these patients. The results suggest that the changed metabolism of phospholipids and protein in liver tissue in cholestatic conditions may contribute to the changes in the serum lipoprotein pattern.     

Usefulness of serum nucleotide pyrophosphatase and phosphodiesterase I activities in classifying liver disease

Nucleotide pyrophosphatase and phosphodiesterase I activities were determined in sera from 126 patients with different types of liver disease and in two additional groups of patients with intra- and extrahepatic cholestasis, respectively. Both activities probably represent the same enzyme, and were positively correlated with alkaline phosphatase, lipoprotein X, and several other tests reflecting cholestasis. Also, we found by discriminant analysis that tests for cholestasis frequently replaced the results of both enzymes. In some groups of liver disease, nucleotide pyrophosphatase and phosphodiesterase I were correlated with the concentrations of prealbumin and albumin. The sensitivity of phosphodiesterase I (and nucleotide phosphatase) is rather low when compared with alkaline phosphatase, and we do not recommend it for use in the clinical routine. Nevertheless, it appears to be of potential value for studies on classification of liver diseases, adding information to a panel of 20 commonly used "liver tests" by appearing in some of the best four test-sets for distinguishing between groups of liver disease by discriminant analysis.     

Plasma alkaline phosphodiesterase I in intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats

1. Administration of alpha-naphthylisothiocyanate (ANIT) to rats produced dose-dependent increases in plasma bile acid and bilirubin concentrations. Similar increases in plasma bile acid and bilirubin concentrations were evident in bile duct ligated rats, indicating that the severity of cholestasis is almost identical in both models. 2. Plasma alkaline phosphodiesterase I was increased by only 50-80% while alkaline phosphatase was increased more than threefold after ANIT administration. This is in contrast to an earlier study [S. R. Simpson, K. Rahman & D. Billington (1984) Clinical Science 67, 647-652] where, after bile duct ligation, serum alkaline phosphodiesterase I was elevated sixfold before any increase in alkaline phosphatase activity became apparent. Thus, plasma alkaline phosphodiesterase I does not offer as sensitive a marker of intrahepatic cholestasis (induced by ANIT) as it does of extrahepatic cholestasis (induced by bile duct ligation). 3. Hepatic alkaline phosphodiesterase I was unaffected by ANIT pretreatment while hepatic alkaline phosphatase was increased up to seven times. It is suggested that raised plasma alkaline phosphodiesterase I is due to regurgitation of the biliary enzyme rather than overspill of the enzyme from liver into blood. 4. Gel filtration showed that 24 h and 96 h after ANIT administration, rat serum contained a high molecular weight form of alkaline phosphodiesterase I, suggesting a different isoenzyme profile.     

The gamma-glutamyltransferase isoenzyme pattern in serum as a signal discriminating between hepatobiliary diseases, including neoplasias

We have used the gamma-glutamyltransferase (GGT) isoenzyme pattern in serum as a means for discriminating between hepatobiliary diseases, including neoplasias. The reference pattern, determined in 142 normal subjects with a simplified conventional cellulose acetate electrophoretic procedure, contained two GGT bands, alpha 1-GGT and alpha 2-GGT, in proportions of 60-80% and 20-40%, respectively. Sera from 95 hepatobiliary patients showed typical isoenzyme features: (a) a beta-migrating GGT form that was less than 10% of the total GGT in chronic hepatitis and cirrhosis, and less than or equal to 30% of the total GGT in cirrhosis with intrahepatic cholestasis and in cases of extra- and intrahepatic obstructive jaundice, including liver neoplasias; (b) a gamma-migrating GGT band and (or) a "dep-GGT" (nonmigrating) band in cases of extrahepatic jaundice; and (c) an albumin-migrating GGT band that had a diagnostic sensitivity of 75% for hepatic tumors. The diagnostic specificity of this last band is 92% toward other hepatic disorders and 91% toward nonhepatic neoplasias; we consider it a potential specific marker for primary or metastatic liver neoplasias.     

Predictive value of assessment of different modalities in the diagnosis of infantile cholestasis

This study investigated the relative accuracy and roles of abdominal ultrasonography, hepatobiliary scintigraphy and liver biopsy in the diagnosis of infantile cholestasis. A total of 50 infants (27 females) aged 1 - 12 months were classified into those with intrahepatic causes of cholestasis (n = 22) and those with extrahepatic causes (n = 28). Cholestasis is caused by a wide range of conditions and diagnosis requires meticulous history taking, thorough clinical examination and many laboratory tests. The most common cause of intrahepatic cholestasis was found to be idiopathic neonatal hepatitis (54.5%), followed by infectious hepatitis (9.1%), metabolic liver diseases (9.1%), intrahepatic biliary atresia (9.1%) and Alagille syndrome (4.5%). The most common cause of extrahepatic cholestasis was extrahepatic biliary atresia (96.4%). The incidence of choledochal cyst was low (3.6%). The cornerstone of the diagnosis of infantile cholestasis was found to be liver biopsy, which was associated with a high degree of accuracy.     

The relevance of some laboratory tests in the diagnosis of cholestasis and the differentiation between intra- and extrahepatic obstruction

The Lp-X test and the determination of total gammaglutamyltransferase activity (GGT), its isoenzyme activities (GGT-1 and GGT-2) and the bile acids have been performed in sera obtained from 66 patients with and without cholestasis. The sensitivity of the Lp-X test as an indicator for cholestasis appeared to be 89% with a specificity of 94%. With the exception of GGT-2 activity, the mean values of total GGT, GGT-1 isoenzyme and bile acids differed significantly between the cholestasis positive and negative groups. The diagnostic use of total GGT, isoenzymes and bile acids in the differentiation between intrahepatic cholestasis and extrahepatic biliary obstruction has been investigated. The GGT-1 isoenzyme activity appeared to be the only parameter differing significantly between the two forms of cholestasis.     

Serum alkaline phosphatase, nucleotide pyrophosphatase, 5'-nucleotide and lipoprotein-X in cholestasis.

The nature of the stationary band of alkaline phosphatase, which occurs on starch gel electrophoresis of sera from patients with biliary obstruction, has been examined. Stationary alkaline phosphatase was eluted from Sepharose 4-B gel close to the void volume and together with the plasma membrane enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, and with lipoprotein-X. Electron microscopy of concentrates of stationary alkaline phosphatase, prepared by ultracentrifugation and gel filtration, showed large (0.3--1 mum diameter) and small structures (30-70 nm diameter) by negative staining. The activity of the stationary alkaline phosphatase was associated in fixed sections with particles of about 10 nm X 40 nm resembling those of lipoprotein-X. It is suggested that the stationary alkaline phosphatase does not move into starch gel during electrophoresis because it is particulate. In agar electrophoresis the alkaline phosphatase which was stationary on starch gel moved towards the cathode with lipoprotein-X.