Accumulation of human apolipoprotein-E in rat plasma after in vivo intramuscular injection of naked DNA.

Naked DNA was found to be incorporated and consistently expressed after in vivo direct injection into striated muscle. In addition to the local expression of muscle-related or exogenous proteins, intramuscular direct gene transfer may be a useful tool to deliver recombinant proteins into the blood stream. However, no direct demonstration of recombinant protein secretion from muscle to the circulation has been reported thus far. We have injected a naked plasmid DNA containing the human receptor-binding defective apo-E2 cDNA, under the control of CMV promoter, into the quadriceps of Yoshida rats, affected by hereditary hypercholesterolemia and altered LDL-receptor activity. Plasma accumulation of human apo-E2 was demonstrated for at least 45 days after injection. On the contrary, the expression of the normal human apo-E3, injected into the muscle of normal Wistar rats, was demonstrated only in the area of muscular injection and not in the blood plasma. Endogenous rat apo-E expression was not affected by the exogenous human apo-E2 production. Our results demonstrate the availability of intramuscular direct gene transfer as a safe and simple method for the chronic systemic delivery of recombinant proteins to the circulation, although further improvements are needed in order to enhance the efficiency and stability of expression.

Effects of disulfiram on serum dopamine-beta-hydroxylase and blood carbon disulphide concentrations in alcoholics.

The aim of this study was to investigate the effects of single and repeated disulfiram doses on serum dopamine-beta-hydroxylase activity and blood carbon disulphide concentrations in a group of abstinent alcoholics. The increase in the blood concentration of carbon disulphide was dose dependent after the oral administration of 100-400 mg of disulfiram. Free carbon disulphide peaked at 12 h while the protein-bound fraction increased at least up to 24 h. Both single (100-400 mg p.o.) and repeated (200 mg daily p.o. for ca. 1 month) administrations failed to inhibit the activity of serum dopamine-beta-hydroxylase. The repeated daily administration of 200 mg of disulfiram also had no influence on copper-activated serum dopamine-beta-hydroxylase, which was the same before and after 1-month treatment period. Contrary to the disulfiram group, the activity of the copper-activated enzyme in the serum of abstinent alcoholics declined significantly during the same 30 days.

Pharmacokinetics of gamma-hydroxybutyric acid in alcohol dependent patients after single and repeated oral doses.

1. The pharmacokinetics of gamma-hydroxybutyric acid (GHB) were studied in 10 alcohol dependent subjects after single and repeated therapeutic oral doses (25 mg kg-1 every 12 h for 7 days). 2. GHB was readily absorbed and rapidly eliminated (tmax = 20-45 min; mean t1/2z 27 +/- 5 s.d. min). Urinary recovery of unchanged GHB was negligible (less than 1% of the dose). gamma-butyrolactone was not detected in either plasma or urine, indicating that lactonization of GHB does not occur in vivo. 3. The multiple-dose regimen resulted neither in accumulation of GHB nor in time-dependent modification of its pharmacokinetics. 4. In five subjects, the data were consistent with nonlinear elimination kinetics of GHB. Administration of a 50 mg kg-1 dose to these subjects resulted in significant increases in dose-normalized AUC, t1/2z and mean residence time. 5. Doubling of the dose also resulted in a significant increase in tmax with little change in Cmax. 6. At the administered doses, GHB did not accumulate in the plasma and caused no serious side effects.

Blood concentration of carbon disulphide in "normal" subjects and in alcoholic subjects treated with disulfiram.

Assay of free and acid labile carbon disulphide (free and total CS2 respectively) in human blood was performed by gas chromatography/spectrometry. The method used a large dynamic head space volume and a "cryogenic trap". Blood CS2 concentration was measured in 42 subjects not occupationally exposed to CS2 (group A) and in 11 alcoholic subjects (group B) treated with disulfiram. Free CS2 concentration showed a mean value of 261 ng/l in the 42 subjects in group A and 9482 ng/l in eight subjects of group B. Total CS2 concentration was 897 ng/l and 40,084 ng/l in groups A and B respectively. Differences between the groups were highly significant for concentrations of both free and total CS2. Total CS2 concentration was about four times as high as free CS2 concentration in both groups. A significant correlation was found between free and total CS2 concentration both in group A and in group B. In the alcoholic subjects (group B), blood concentrations of both free and total CS2 were related to time of sampling after treatment with disulfiram.

Diagnostic and prognostic value of the determination of the aminopropeptide of type III procollagen in patients with primary liver cancer.

Hepatic fibroplasia seems to play an important role in the course of primary liver cancer (PLC) since, for instance, encapsulated and fibrolamellar hepatocellular carcinomas show a definitely better prognosis. In this study, serum procollagen III amino-terminal peptide (PIIIP) levels, which reflect synthesis and release of procollagen type III, were measured with the aim of assessing hepatic fibrogenesis in PLC patients and determining whether serum PIIIP levels play a diagnostic or prognostic role in PLC. Twenty-five patients with PLC, 74 patients with cirrhosis and 38 healthy volunteers were studied. Serum PIIIP levels were determined by a radioimmunoassay (RIA) method. In PLC patients PIIIP serum levels were significantly higher than those of controls and cirrhotic patients (P less than 0.001 and P less than 0.01 respectively) but an analysis of individual values showed an important overlap between PLC and cirrhosis. No correlation was found between serum PIIIP levels and tumour histology, presence or absence of cirrhosis, Child status, possible aetiology of the disease, indices of hepatocellular inflammation, cholestasis and synthesis, or tumour markers. On the contrary, serum PIIIP levels correlated with tumour gross pattern (z = 3, P less than 0.001) and, inversely, with survival (r = 0.659, P less than 0.01), patients with serum PIIIP over 25 ng/mL showing a significantly worse prognosis. These data confirm that hepatic fibroplasia plays an important, but not yet fully understood, role in the course of PLC. From the clinical point of view, PIIIP determination does not add to the differential diagnosis between PLC and cirrhosis but helps to identify patients with larger liver replacements and worse prognoses.

[Changes in blood Zn, Se, Cd, Pb during therapeutic abstinence from alcohol in 101 cases of alcoholism]. / Variazioni ematiche di Zn, Se, Cd, Pb durante astinenza alcolica terapeutica in 101 casi di alcolismo.

The patients were subdivided into 3 groups, in accordance with clinical, echographic, laboratory and laparoscopy criteria: without liver disease, with liver disease and with alcoholic cirrhosis. In alcoholics with liver disease (with or without cirrhosis), we found a constant significant decrease in lead during abstinence, while in patients with cirrhosis zinc has a significant increase. Always lower to normal the blood selenium. The cadmium in the serum increases also after a month.

[Evaluation of the sexual role in a group of alcoholics]. / Valutazione del ruolo sessuale in un gruppo di alcolisti.

The sexual role, evaluated by administration of the Bem Sex Role Inventory in 33 alcoholics, proved significantly different from that seen in a typical Italian sample and that presented by subjects suffering from other organic pathology. No significant differences were observed in psychic symptomatology between alcoholics with sexual role of androginous type with respect to the non-androginous.

Sex hormones and sex hormone binding globulin in males with compensated and decompensated cirrhosis of the liver.

The present work investigates the sex hormone profiles in 50 male patients with liver cirrhosis of different etiology according to the degree of liver dysfunction. The only hormonal impairment in well-compensated cirrhotics (group A) was an increase in mean serum concentrations of estrone, androstenedione, and sex hormone binding globulin. In decompensated cirrhotic patients with ascites (group B), low mean levels of total and free testosterone were found along with normal gonadotropins mean levels. Estrone and androstenedione levels were still elevated, whereas sex hormone binding globulin levels were not different from controls. In decompensated cirrhotics patients with encephalopathy (group C), total and free testosterone mean levels were lower than in group B, and LH mean levels were elevated; estrone levels were markedly high, but androstenedione levels were subnormal; sex hormone binding globulin concentrations were again not different from controls. The few patients with high prolactin levels belonged primarily to this group. Estradiol mean levels were not significantly elevated in any of the groups. It is concluded that the various hormonal patterns of gonadal failure and of the impairment of steroid metabolism and transport, observed in cirrhosis, can be attributed to the degree of liver dysfunction.

Relationships between blood lead concentration and aminolevulinic acid dehydratase in alcoholics and workers industrially exposed to lead.

Blood lead concentration (Pb-B), aminolevulinic acid dehydratase (ALAD), and gamma-GT were measured in 265 workers industrially exposed to lead and in 184 patients with liver disease resulting from alcohol consumption. The first group was divided according to alcohol use, i.e., nondrinkers, moderate drinkers, and heavy drinkers. The second group was divided according to the following criteria: hepatopatic without cirrhosis, hepatopatic with compensated cirrhosis, and hepatopatic with decompensated cirrhosis. Heavy drinkers who were industrially exposed had the highest Pb-B (40.4 +/- 14.6 micrograms/dl) and the lowest ALAD (22.2 +/- 9.1 U/L). The correlations between Pb-B and ALAD show no significant change with the increase of Pb-B. In the alcoholic group, 76 patients with alcoholic liver disease without cirrhosis had the highest Pb-B (40.3-9.1 micrograms/dl) and ALAD the lowest (18.6 +/- 7.7 U/L). The negative correlation between Pb-B and log ALAD disappeared completely in individuals with Pb-B that exceeded 50 micrograms/dl, independent from the seriousness of illness.

Primary and metastatic tumors of the liver associated with cirrhosis. A study based on laparoscopy and autopsy.

In a series of 2,538 cases of cirrhosis seen at laparoscopy there were 140 primary liver carcinomas and 19 cases of metastases to a cirrhotic liver out of a total of 167 extrahepatic neoplasms associated with cirrhosis. In an autopsy series of 1,073 cases of cirrhosis there were 190 primary liver carcinomas and 22 cases of metastases to a cirrhotic liver out of a total of 98 extrahepatic neoplasms. In another autopsy series of 498 cases of cirrhosis there were 71 primary liver carcinomas and 18 cases of metastases to a cirrhotic liver out of a total of 58 extrahepatic neoplasms. The laparoscopy series showed a predominance (31.8%) of esophageal tumors associated with cirrhosis, but these tumors rarely gave rise to liver metastases (3.7%); in the autopsy series there was a predominance (35.3%) of tumors of the portal territory, giving rise to metastases in cirrhotic livers in 35.2% of cases.

Discriminant analysis in the clinical and biochemical diagnosis of primary liver cancer.

Discriminant analysis was used in evaluating the importance of clinical aspects and the value of routine and experimental biochemical markers in the differential diagnosis of primary liver cancer (PLC) and chronic, non-neoplastic, liver diseases. Our results show that: 1) Clinical signs, such as the presence of pain, weight loss or mass, correctly indicate the diagnosis in 76% of the cases; 2) The determination of alkaline phosphatase isoenzymes is shown by the computer to be the most useful marker and provides an overall diagnostic accuracy which is higher than that of alpha-fetoprotein. We also found that, by using these two markers together, "by intersection," the best overall accuracy (85%) is obtained. We, therefore, suggest determination of alkaline phosphatase isoenzymes and alpha-fetoprotein in screening the populations at risk for liver cancer.

Laparoscopy in the diagnosis of primary carcinoma of the gallbladder. A study of 98 cases.

Ninety-eight cases of carcinoma of the gallbladder were studied by laparoscopy from 1968 to 1982. The gallbladder was completely explored in 48 patients. Hard white plaques of the gallbladder wall were noted in 30 patients. Local metastases were found in 89 patients. A diagnosis of malignancy by biopsy was made in 90% of cases although only three biopsies were taken from the gallbladder itself. Laparoscopy is useful in identifying those rare cases where radical surgical intervention may improve survival.

Detection of immunoglobulins G and A on the cell membrane of hepatocytes from patients with alcoholic liver disease.

The presence of immunoglobulins (Ig) G, A, and M and of complement fractions (C3-C4) on the liver cell surface was investigated by direct immunofluorescence in 40 patients with alcoholic liver disease. IgG was detected on the liver cell membrane with a linear staining pattern in 29 patients. The percentage of IgG-positive hepatocytes correlated with transaminase activities, independently of the histological findings. IgA was demonstrable with a coarse granular staining pattern in 11 of the 14 cases with established cirrhosis. The finding of IgG bound to the hepatocyte surface in patients with alcohol-induced liver damage suggests that alcohol could be responsible for antigenic modifications of hepatocyte membrane with consequent triggering of a humoral immune response.

Blood-lead levels in patients with chronic liver diseases.

Blood-lead concentrations (Pb-B) were measured in 318 adult inpatients with chronic liver diseases. The Pb-B was highest (387 +/- 96 micrograms/l) in 102 patients with alcoholic liver disease without cirrhosis. The Pb-B was still high, but significantly lower in 60 patients with compensated alcoholic cirrhosis (342 +/- 100 micrograms/l) and in 72 patients with decompensated alcoholic cirrhosis (312 +/- 97 micrograms/l). This difference was in part due to a significant decrease of the hematocrit which fell from 44.4 +/- 4.9% to 42.4 +/- 27.2% and to 39.2 +/- 7.4% respectively. In patients with viral or cryptogenic liver diseases the Pb-B was 211 +/- 69 micrograms/l in 11 patients with chronic persistent hepatitis, 219 +/- 72 micrograms/l in 19 with chronic active hepatitis, 206 +/- 94 micrograms/l in 28 with compensated cirrhosis, and 226 +/- 98 micrograms/l in 26 with decompensated cirrhosis, without any significant difference. The Pb-B of the male patients showed no correlation to age, with the exception of 25 male patients with chronic persistent and active hepatitis (r = 0.626, P less than 0.001).

[At II in liver diseases. Comparison between immunological determination and that with chromogenic substrate]. / L'AT III in patologia epatica. Confronto tra la determinazione immunologica e quella con substrato cromogeno.

Plasma antithrombin III activity determined as a heparinic cofactor, by means of chromogenic substrate (Cromozym TH) has been compared with protein concentration of AT III (radial immunodiffusion) in patients with a variety of liver conditions. Reference values for AT III were obtained from the plasma of 50 donors whose state of health was confirmed by simultaneous determination of 20 haematochemical parameters (SMAC-Technicon). The patients were classified according to clinical, laparobioptic and laboratory data and put through a series of clotting tests including PT, fibrinogen, FDP, Hepatoquick, Platelet count. In healthy donors, the activity and protein concentration of antithrombin III were interrelated, as they were even in cirrhotic patients (r = 0.77) both being markedly reduced; in chronic hepatitis, diminution with both methods was modest and correlation less apparent (r = 0.48).

[Sulfobromophthalein clearance and survival of cirrhotics after an elective porto-systemic shunt]. / Clearance della bromosulfonftaleina (B.S.F.) e sopravvivenza dei cirrotici dopo uno shunt porto-sistemico di elezione.

Preoperative clearance of a single dose of BSP was assessed in 72 candidates for porto-systemic shunt in cirrhosis of the liver. Comparison between those who had died (24) and those who had survived (48) several years later showed statistically significant differences with regard to equivalent liver volume, first uncorrected exponential (Ki), absolute clearance, and 45' retention. A certain degree of differentiation could be made out among the survivors, whereas those who had died could not be distinguished from the series as a whole, nor could correlations be established with the length of survival. No practical advantage was displayed by the more complex, computer-processed parameters as opposed to those that were more immediately available.

[Changes of the biohumoral picture in alcoholic hepatopathy treated with thiazolidinecarboxylate of arginine]. / Variazioni del quadro bioumorale della epatopatia etilica trattata con tiazolidincarbossilato di arginina.

In 28 patients suffering from alcoholic well compensated hepatopathy, different laboratory parameters have been controlled before and after treatment with arginine thiazolidinecarboxylate, i.v. a the daily dose of 800 mg for 7-10 days. The compartmental analysis showed that the improvement in the BSF clearance is mainly due to a reduction in the "regurgitation fraction" and therefore to an improvement or restoration of the processes of conjugation, binding and transferring of the dye in the liver cells. A significant increase in platelets and an equally significant decrease in bilirubinemia, gamma GT, OCT, GOT and alkaline phosphatase were reported. Such variations might be at any rate due to the sole alcohol abstinence. The observed increase in transferrin, haptoglobin and albumin cannot be reported to the sole abstinence, but must be considered as consequence of the pharmacological influence, of arginine thiazolidinecarboxylate on the protidosynthetic activities of the liver.