Serum alpha fetoprotein heterogeneity as a means of differentiating between primary hepatocellular carcinoma and hepatic secondaries

The concanavalin A binding characteristics of serum alpha-fetoprotein (AFP) were investigated in patients with primary hepatocellular carcinoma and hepatic secondaries using affinity column chromatography and radioimmunoassay. The primary hepatocellular carcinoma (n = 21) was associated with a median concanavalin A non-reactive AFP fraction of 7.4% (range 1.6 - 18.8) while the hepatic secondaries (n = 8) had a median concanavalin A non-reactive AFP fraction of 50.7% (range 26.6 - 91.7). A simple diagnostic test for differentiating between the two groups of patients is proposed.     

Increased elastase-alpha 1-antitrypsin complex in fulminant hepatic failure: relationship to bacterial infection and activation of coagulation.

To study the effect of infection, a frequent complication of fulminant hepatic failure (FHF), on the release of elastase from polymorphonuclear leucocytes and its inhibition in circulation we have measured the concentrations of alpha 1-antitrypsin, which binds and inhibits elastase in the circulation, and of elastase-alpha 1-antitrypsin complex, in 30 patients with FHF. Elastase-alpha 1-antitrypsin complex was significantly increased in FHF as compared to controls (303 +/- 51 micrograms/l compared to 37 +/- 5 micrograms/l; n = 10; P less than 0.001) demonstrating activation of leucocytes in FHF. Infection caused greater release of leucocyte elastase, complex levels were significantly greater in patients who were infected when compared to those who were not (463 +/- 84 micrograms/l; n = 13 compared to 180 +/- 46 micrograms/l; n = 17; P less than 0.01). Also patients who survived had significantly lower complex levels than those who did not (212 +/- 49 micrograms/l; n = 18 compared to 440 +/- 94 micrograms/l; n = 12; P less than 0.02). alpha 1-Antitrypsin activity was not significantly different from control subjects (0.99 +/- 0.06 U/ml compared to 0.97 +/- 0.05 U/ml). However alpha 1-antitrypsin activity was significantly higher in patients who survived (1.17 +/- 0.05 U/ml; n = 18) compared to those who did not (0.71 +/- 0.03 U/ml; n = 12; P less than 0.001) and patients who died had significantly lower levels than control subjects (P less than 0.01) indicating the importance of maintenance of normal inhibitor levels in patients with FHF. The leucocyte activation and release of elastase in FHF was linked to activation of the coagulation system; elastase--alpha 1-antitrypsin complex levels correlated significantly with thrombin-antithrombin III complex levels (r = 0.68; P less than 0.001) and inversely with fibrinogen (r = -0.71; P less than 0.001).     

The role of the false neurotransmitter octopamine in the hypotension of fulminant hepatic failure.

1. An investigation was carried out into the mechanism of unexplained hypotension in patients with fulminant hepatic failure. The cardiac output and peripheral resistance were compared in normotensive and hypotensive patients. In addition, the serum concentration of the false neurotransmitter octopamine and the pressor response to noradrenaline, and to the indirectly acting sympathomimetic agent tyramine, were measured in hypotensive and normotensive patients with fulminant hepatic failure and in healthy subjects. 2. The cardiac output and the peripheral resistance were decreased in the hypotensive patients, and their mean heart rate was slower than in the normotensive patients. Although the serum octopamine concentration was significantly elevated in the patients compared with the control subjects, the highest octopamine concentrations were unexpectedly found in the normotensive patients and a significant positive correlation could be demonstrated between the resting blood pressure and the serum octopamine concentration. The pressor response to tyramine and noradrenaline were similar in the hypotensive patients, the normotensive patients and control subjects. 3. These results suggest that neither increased serum concentrations of the false neurotransmitter octopamine, nor end-organ insensitivity to released noradrenaline are responsible for the hypotension. A more likely explanation is toxic depression of the vasomotor centre. The opening of peripheral arteriovenous shunts, possibly as a result of endotoxaemia, might be an additional factor.     

The microheterogeneities of thyroxine-binding globulin and alpha 1 protease inhibitor (alpha 1-antitrypsin) are interrelated

Addition of 125I-thyroxine to serum allows autoradiography for thyroxine-binding globulin microheterogeneity to be carried out after isoelectric focusing has been performed to display (by protein stain) the heterogeneous bands of the alpha 1-antitrypsin (PI) system. Comparison of the protein stain for PI with the autoradiograph for thyroxine-binding globulin indicates that these two systems are interrelated with the major bands of the PI system corresponding to the bands on the autoradiograph. This correspondence holds for PI variants other than the common M type and in particular it holds for the deficient Z type in which the autoradiograph for thyroxine-binding globulin is strikingly different from normal. We conclude that the major cause of microheterogeneity of TBG is due to an association with the PI system under the conditions of isoelectric focusing as normally performed. Precipitation experiments with antisera to PI and TBG suggest that the complex between these biologically important globulins may occur under conditions other than isoelectric focusing, but further work will be needed to examine this possibility.     

Thyroxine Turnover and Transport in Laennec's Cirrhosis of the Liver

Studies of the metabolism of thyroxine in 14 cases of cirrhosis revealed a variety of deviations from normal. In addition to radiothyroxine turnover studies, determinations were made of the free thyroxine fractions and free thyroxine iodine concentrations in serum (magnesium precipitation method) as well as the maximal binding capacities of thyroxine-binding alpha globulin (TBG) and thyroxine-binding prealbumin (TBPA) by reverse flow paper electrophoresis in a glycine acetate system at pH 8.6.     

alpha 2-Macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency

Serum alpha 2-macroglobulin concentrations were measured in 178 patients with emphysema and 115 control subjects of similar age and sex distribution. The study group included 59 PI type Z patients with alpha 1-antitrypsin deficiency, five with the rare alpha 1-antitrypsin null genotype (PI Q0 or --), and seven with alpha 1-antitrypsin deficiency of the rare PI types MmaltonZ or MduarteZ. Individuals with all types of alpha 1-antitrypsin deficiency were found to have significantly increased serum concentrations of alpha 2M (p less than 0.001). These increased concentrations were associated with all types of alpha 1-antitrypsin deficiency, not only with the PI type Z. The highest alpha 2-macroglobulin concentrations were found in the PI Q0 patients (5 with emphysema, 2 with no lung disease), and these patients had almost no circulating alpha 1-antitrypsin. Raised concentrations of serum alpha 2-macroglobulin were not due to emphysema: 86 patients with emphysema, of PI type M, and the normal control subjects had similar average concentrations of alpha 2-macroglobulin. One control subject with an average alpha 2-macroglobulin concentration of only 41% of normal was found.     

Evaluation of serum triiodothyronine and adjusted triiodothyronine (free triiodothyronine index) in pregnancy.

We measured serum thyroxine (free and total), triiodothyronine (free and total), thyroxine-binding globulin, and triiodothyronine uptake by talc in 97 normal men and 50 pregnant women. Mean serum thyroxine and triiodothyronine concentrations were higher in the pregnant subjects (104 vs. 78 mug/liter and 1.69 vs. 1.30 mug/liter) because of a higher mean thyroxine-binding globulin concentration (70 vs. 38 mg/liter). Mean triiodothyronine uptake by talc was lower in the pregnant subjects (0.82 vs. 1.03). Mean free thyroxine concentrations were similar in the two groups, but mean free triiodothyronine concentrations were 10% lower in the pregnant subjects. Triiodothyronine uptake by talc and the diayzable thyroxine and triiodothyronine fractions were highly correlated (r = 0.85 and r = 0.82, P less than 0.001). Calculated free thyroxine index and free triiodothyronine index values (hyroxine and triiodothyronine indirectly adjusted, using triiodothyronine talc uptake to compensate for differences in thyroxine-binding globulin concentration), were statistically similar (84 vs. 82 and 1.38 vs. 1.34) in pregnant and male subjects. The results indicate that the total triiodothyronine concentration can be normalized on the basis of the triiodothyronine uptake by talc to correct for variations in thyroxine-binding globulin concentration.     

Serum octopamine, coma, and charcoal haemoperfusion in fulminant hepatic failure

Serum octopamine levels were significantly higher in twenty patients with fulminant hepatic failure (FHF) during the first 48 h of grade IV coma than in health control subjects (3.38 +/- 0.20 ng/ml and 1.75 +/- 0.19 ng/ml respectively, P less than 0.001). Serial measurements in five patients who died without regaining consciousness showed serum octopamine to remain raised, and concentrations in the cerebrospinal fluid at death reflected serum levels. In five patients who regained consciousness, improvement in encephalopathy was associated with a significant reduction in serum octopamine. Renal failure in patients with FHF was found to contribute to raised serum octopamine but could not alone account for the observed levels. Patients given neomycin therapy did not have significantly lower serum octopamine levels than an untreated group. There was, however, a significant correlation between elevated serum octopamine and the occurrence of gestrointestinal bleeding during the previous 24 h. Charcoal haemoperfusion did not appreciably reduce serum octopamine levels.     

Elevated serum concentrations of thyroxine-binding globulin and caeruloplasmin in methadone-maintained patients.

Patients on a methadone-maintenance program had high serum concentrations of thyroxine-binding globulin. This resulted in elevated serum thyroxine and triiodothyronine levels and low triiodothyronine uptake values. Their free thyroxine index was normal, as was serum thyroxine and triiodothyronine when related to thyroxine-binding globulin concentration. Serum caeruloplasmin concentration was also increased in methadone-treated patients.     

A new radioimmunoassay of free thyroxine using I-labelled thyroxine—protein complex uninfluenced by albumin and thyroxine-binding globulin

We describe a double-antibody solid phase radioimmunoassay for free thyroxine (FT₄) in serum with use of ¹²⁵I-labelled thyroxine-human chorionic gonadotropin conjugate. Since the labelled conjugate does not bind to thyroxine binding globulin (TBG) and albumin because of its large molecular weight, the method is uninfluenced by TBG or albumin. The measurable range of FT₄ in serum was 2.0 to 128 ng/l. The mean coefficients of variation within and between assays were 4.6–8.6% and 6.3–11.6%, respectively. The FT₄ values determined by the proposed method correlated well with those determined by commercial radioimmunoassay in subjects with normal albumin concentration (r = 0.98). The FT₄ concentrations in serum as determined by this method were 9 to 17 ng/l for healthy adult subjects; high for patients with hyperthyroidism; low for patients with hypothyroidism; and within normal limits for pregnant women, and patients with high or low concentrations of thyroxine-binding globulin.     

An evaluation of stabilizing agents in competitive protein-binding assay for 25-hydroxyvitamin D

We have recently described a major variation from the normal levels of serum thyroxine-binding globulin in many Australian Aborigines [1]. Subsequently we presented evidence that affected individuals were widely distributed throughout Australia, that “low” values of thyroxine-binding globulin were not caused by environmental or health factors, but were inherited in an autosomal dominant fashion [2]. Refetoff [3] has shown that the cause of genetically determined low thyroxine-binding globulin levels in Caucasians is alteration in synthesis rate without any structural variation of the protein. Since however the “low” thyroxine-binding globulin of Aborigines is vastly more prevalent and genetically distinct from the X-linked type, we investigated the possibility that this may be a structural variant. Evidence suggestive of this includes results from heat inactivation, competitive binding of thyroxine to thyroxine-binding globulin to measure affinity, and use of a radioimmunoassay different from that used in the original work. The “low” thyroxine-binding globulin of Aborigines may be a protein with a structural variation at or near the binding site for thyroxine, resulting in lower affinity for thyroxine and hence “low” results with assay methods which depend upon the thyroxine binding site. Since the Australoid peoples, to whom Australian Aborigines are racially related, are distributed widely throughout the southern hemisphere it is important to establish whether this variant is found outside Australia in order to avoid the likelihood of misdiagnosis of thyroid disease in such subjects.     

Nephelometry of acute-phase glycoproteins by binding to concanavalin A

Nephelometry of serum acute-phase glycoproteins by binding to concanavalin A (con-A) was compared with assays for haptoglobin and alpha 1-acid glycoprotein, and for C-reactive protein. The cutoff points for positive reactions were determined on the basis of results for a random sample (n = 130) from a middle-aged population. The sensitivity of the con-A binding assay compared favorably with that of individual acute-phase glycoproteins in a follow-up cohort of 198 patients with inflammatory joint diseases. Unlike the case in many individual acute-phase glycoprotein assays, the distribution of con-A binding values in healthy subjects is remarkably symmetrical, allowing an easy distinction between abnormal and normal values.     

Microheterogeneity of serum glycoproteins in patients with chronic alcohol abuse compared with carbohydrate-deficient glycoprotein syndrome type I.

BACKGROUND: Chronic alcohol abuse alters the normal N-glycosylation of transferrin, producing the carbohydrate-deficient transferrin isoforms. This alteration could be similar to that present in patients with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1). We thus compared the alterations of N-glycans present in patients with alcoholism and patients with CDG1. METHODS: The N-glycans of serum glycoproteins were compared in sera of patients with alcoholism, patients with CDG1, and controls by two-dimensional electrophoresis, neuraminidase, peptide:N-glycosidase F, and endoglycosidase F2 treatments. A specific antibody directed against the amino acid sequence surrounding the N-432 N-glycosylation site of transferrin was prepared (SZ-350 antibody). RESULTS: In patients with alcoholism, the abnormal transferrin and alpha(1)-antitrypsin isoforms were devoid of a variable number of entire N-glycan moieties and were identical with those present in CDG1. In the serum of patients with alcoholism, this finding was less pronounced than in CDG1. In contrast to CDG1, there was no decrease in clusterin or serum amyloid P in patients with alcoholism. The SZ-350 antibody recognized only transferrin isoforms with one or no N-glycan moieties. CONCLUSION: Antibodies directed against specific N-glycosylation sites of glycoproteins could be useful for developing more specific immunochemical tests for the diagnosis of chronic alcohol abuse.     

Concentrations of protease and anti-protease in serum of patients with pancreatic cancer

We measured the concentrations of trypsin, elastase, and three anti-proteases-alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin-in serum from 10 patients with pancreatic carcinoma. All 10 showed increased elastase and decreased alpha 2-macroglobulin concentrations, nine had increased alpha 1-antichymotrypsin, and eight had increases in alpha 1-antitrypsin and trypsin. Serial studies during chemotherapy of one patient showed that the protease concentrations decreased during treatment but the concentrations of the anti-proteases remained abnormal.     

Energy expenditure in acetaminophen-induced fulminant hepatic failure

OBJECTIVE: To determine energy expenditure in critically ill patients suffering from acetaminophen-induced fulminant hepatic failure and compare it with values obtained in matched, healthy control subjects and in patients studied during the anhepatic period of elective liver transplantation. DESIGN: Prospective, controlled, observational study. SETTING: A ten-bed intensive therapy unit and a liver transplant unit at a University teaching hospital. PATIENTS AND SUBJECTS: Sixteen patients suffering from acetaminophen-induced fulminant hepatic failure who were sedated, paralyzed, and mechanically ventilated; 16 age-, gender-, and weight-matched, awake, healthy control subjects; and 16 patients with chronic liver disease, undergoing elective liver transplantation, who were studied during the anhepatic period of surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The mean energy expenditure was calculated in each case for a 30-min period, using indirect calorimetry. In the patients undergoing liver transplantation, measurements were performed after clamping the hepatic veins and recipient hepatectomy. Energy expenditure was markedly increased in the fulminant hepatic failure group (mean energy expenditure, 4.05 [SD 0.52] kJ x kg(-1) x hr(-1)), in comparison with healthy control subjects (mean, 3.44 [0.27] kJ x kg(-1) x hr(-1); mean difference, 18%; p < .001) and in comparison with patients during the anhepatic period of liver transplantation (mean, 3.15 [0.61] kJ x kg(-1) x hr(-1); mean difference, 29%; p < .001). These differences were even more pronounced when a correction factor for differences in core temperature was included in the calculation. Harris-Benedict predictions of energy expenditure were unreliable in the patients with acute liver failure. No correlations were found among energy expenditure and hemodynamic variables, the requirement for vasoconstrictors, or the presence of renal failure. CONCLUSIONS: Despite the loss of functioning liver cell mass, the metabolic rate is substantially increased in patients with acetaminophen-induced fulminant hepatic failure. This finding is consistent with the marked systemic inflammatory response, which accompanies acute hepatic failure. The Harris-Benedict equation is unreliable when an estimation of energy expenditure is required in patients with this condition.     

Affinity chromatographic separation of alpha-fetoprotein variants: development of a mini-column procedure, and application to cancer patients

This is an affinity chromatographic procedure for separating variants of alpha-fetoprotein in 0.1 mL of serum on a disposable mini-column containing 0.9 mL of concanavalin A-Sepharose, at pH 6.4 and with a flow rate of 5 mL/h. The binding capacity of the column is 52 micrograms. Analytical recovery was 90%. Between-run CVs varied from 2.0% for samples with a high percentage of concanavalin A nonreactive fraction to 13% for samples with a low percentage. The mean proportion of nonreactive alpha-fetoprotein was 13.4% for patients with hepatocellular carcinoma, 62.2% for testicular carcinoma, and 8.9% for nonmalignant liver diseases. This suggests that the alpha-fetoprotein variants could be used to distinguish hepatocellular carcinoma from other carcinomas. Serial determination of the concanavalin A nonreactive fraction in such patients showed a relatively constant percentage. This indicates that monitoring variants in patients with hepatocellular carcinoma throughout their clinical course may not provide useful information additional to that provided by the values for total serum alpha-fetoprotein.     

A simple ligand-binding assay for thyroxine-binding globulin on reusable Sephadex columns.

A method for the assay of thyroxine-binding globulin on reusable Sephadex G-25 (fine) columns is described. It depends upon elution by diluted iodothyronine-free serum of protein-bound [125 I]thyroxine from the columns under conditions where binding to thyroxine-binding prealbumin and albumin are abolished. It is simple, rapid and precise and permits determinations in large numbers of samples. Values (mg/l; mean +/- S.D.) were: normals 31.6 +/- 5.4, hyperthyroid 28.3 +/- 4.8, hypothyroid 40.6 +/- 7.5, oral contraceptives 40.1 +/- 6.8, pregnant 50.3 +/- 5.4, cirrhotics 20.7 +/- 4.3. Concentrations were reduced in serum heated at 56 degrees C, while the uptake of [125 I]triiodothyronine was increased. There was a significant negative correlation between thyroxine-binding globulin concentration and triiodothyronine uptake in the heated serum samples and in euthyroid subjects.     

Interference of iodine-125 ligands in radioimmunoassay: evidence implicating thyroxine-binding globulin.

Although there is abundant published evidence that radioiodinated antigens interfere in digoxin radioimmunoassays, other radioimmunoassays are similarly affected. We investigated the relationship of radioiodinated antigen structure to its function in the immunoassay. Carrier-free 125I-labeled iodotyrosine and iodohistamine derivatives were incubated with human serum, and the bound and free fractions were separated. We demonstrated that diiodotyrosyl analogs bind avidly to serum proteins. Because protein binding could be reduced with competitors that inhibit thyroxine-binding globulin, such as 1,8-anilinonaphthalene sulfonate and thyroxine, thyroxine-binding globulin was clearly implicated. Diiodotyrosyl compounds also bound to solutions of purified thyroxine-binding globulin, and this binding was inhibited by the same two competitors. We postulate that thyroxine-binding globulin is the major source of the heretofore unexplained interference of radioiodinated haptens. We present recommendations for eliminating or minimizing such interference.     

Relationship between serum sialic acids, sialic acid-rich inflammation-sensitive proteins and cell damage in patients with acute myocardial infarction.

The role of sialic acid (SA) in the pathogenesis of atherosclerosis and as a predictor of cardiovascular events has attracted much attention in recent years. However, most studies investigating the role of total and lipid-bound sialic acids (TSA and LSA) in the pathogenesis of atherosclerosis lack information on the reason for the elevated SA concentrations in coronary heart disease and myocardial infarction. Since the inflammation-sensitive proteins are glycoproteins with SA residues, an increase in their levels due to some type of acute-phase reaction or inflammation could be responsible for the elevated TSA levels in acute myocardial infarction (AMI). Elevated serum SA levels might also be due to either shedding or secretion of free SA from the cell or cell membrane surface if neuraminidase levels are increased, or to the release of cellular SA-containing glycolipids and/or glycoproteins into plasma from myocardial cells after AMI. The aim of the present study was to investigate both the possible role of SA-rich inflammation-sensitive proteins and the cell damage due to elevated serum TSA levels in AMI. A possible role of serum LSA as an indicator of the shedding or secretion of SA from the cell or cell membrane surface in AMI was also evaluated. The study included 38 subjects with AMI and 32 healthy volunteers. Serum TSA and LSA were determined using the methods of Warren and Katopodis, respectively. The concentrations of serum SA-rich inflammation-sensitive proteins, namely alpha1-antitrypsin, alpha2-macroglobulin and ceruloplasmin were determined immunoturbidimetrically. Our data showed that: a) mean levels of serum TSA and LSA and SA-rich inflammation-sensitive proteins in patients with AMI were significantly increased; and b) there was a significant positive correlation between TSA and LSA and alpha1-antitrypsin in patients with AMI. Since the transfer of free SA to lipoproteins is required for an increase in serum LSA levels, and free SA for this transfer can be provided by the secretion of SA from the cell, it is obvious that the shedding or secretion of SA from the cell membrane surface or release of cellular SA from cells into the bloodstream due to cell damage after AMI also occur after AMI. As a result, we can report that either the shedding or secretion of SA from the cell or cell membrane surface and the increased output of SA-rich inflammation-sensitive proteins may together be responsible for the elevated TSA levels in AMI.     

Glycoproteins of pleural effusions (author's transl)]

Using single radial immunodiffusion, ten glycoproteins from non purulent pleural fluids have been estimated in different diseases. For five proteins (prealbumin, ceruloplasmin, alpha2HS-glycoprotein, transferrin, beta2-glycoprotein 1) the results have been found not to correlate with the causal disease. However for orosomucoid, alpha1-antitrypsin, haptoglobin, alpha2-macroglobulin and hemopexin, there was good correlation between proteins levels and aetiology. The glycoprotein concentration was low in mechanical effusions from cirrhosis and chronic cardiac failure. It was high in inflammatory, post-embolism and particularly neoplastic effusions. A raised orosomucoid level occurred as the most characteristic of cancer states especially when associated with a parallel increase of the four other glycoproteins. A simultaneously elevated level of these five pleural glycoproteins seems to be a good and significant biological sign for neoplastic effusion diagnosis.