Development of severe alcohol related liver disease over four decades in Iceland: impact of increased access and use of alcohol.

OBJECTIVE: Limited data exist on the association between per capita alcohol consumption and incidence of alcohol related liver disease (ARLD). The aims were to analyse this relationship and assess prevalence of ARLD in Iceland and among patients treated for alcohol use disorder (AUD) and its impact on outcomes. METHODS: A retrospective study on all patients diagnosed with severe ARLD: alcohol related cirrhosis (ARC) and alcohol related hepatitis (ARH) in Iceland 1984-2020. Medical records were scrutinized for clinical features, severity of ARLD, proportion undergoing treatment for AUD, data on abstinence and long-term outcomes. RESULTS: A total of 314 patients, males 76%, median age 56 years, fulfilled the predetermined criteria for ARLD. Median MELD was 17, 73% with Child-Pugh B/C and 70/314 (22%) who had ARH. Incidence of ARLD increased from 0.77 cases per 100 000 inhabitants annually 1984-2000 to 6.1 per 100 000 in 2016-2020. Per capita alcohol consumption increased from 4.3 Liters to 7.5 L in in the same time periods. Overall 220/314 (70%) with ARLD had undergone treatment for AUD. Of all individuals who had AUD treatment during the study period (n = 21.845), 1% were diagnosed with ARLD. Patients who underwent treatment for AUD after the ARLD diagnosis had better prognosis than those who had treatment prior to ARLD diagnosis (hazard ratio 2.5 [95% CI 1.3-5.0]). CONCLUSIONS: The incidence of ARLD increased 8-fold during the study period coinciding with 74% increase in per capita alcohol consumption. Patients with prior diagnosis of AUD had worse prognosis that needs special attention.

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) for the detection of picogram quantities of IgE.

Existing methods of measuring IgE in in vitro peripheral blood lymphocyte (PBL) cultures are not sufficiently sensitive to detect IgE when it is present in small amounts. This paper describes a modification of a two-site ELISA which increases the sensitivity of the assay 10-20-fold. By using the Fab' fragment of either rabbit or mouse monoclonal anti-IgE conjugated to alkaline phosphatase (AP) as the detector, the background of the assay was reduced sufficiently to permit signal amplification, using a commercially available amplified AP substrate. With this assay as little as 10 pg/ml of IgE could be detected. The interassay coefficient of variation was 15-18% between 1200 and 100 pg/ml IgE (n = 14) and there was a good correlation with a commercial IgE radioimmunoassay (RIA) (r = 0.98, n = 38).

Enzyme-amplified immunoassays.

The sensitivity of enzyme immunoassays may be enhanced by the use of enzyme-amplification. This technique uses the enzyme label in the immunoassay to provide a trigger substance for a secondary system that can generate a large quantity of coloured product. Two examples of enzyme amplifiers are described, using either a substrate cycle with phosphorylated hexose sugars, or a redox cycle involving the coenzyme NAD(+). The redox enzyme-amplifier has a detection limit of less than one attomole for the enzyme label, alkaline phosphatase. The limited dynamic range of enzyme-amplified immunoassays may be overcome by kinetic analysis of the colour development in the enzyme-amplifier, to add at least a further order of magnitude to the range of directly measured analyte concentrations in the immunoassay. This is illustrated in an enzyme-amplified immunoassay for human thyroid stimulating hormone. Amperometric measurement of the enzyme-amplifier provides a method to extend the dynamic range still further and compares favourably with the performance of a gamma counter, a luminometer or a fluorimeter.

Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole.

A method is described for increasing the response of enzyme immunoassays employing alkaline phosphatase as the label initiating 2 sequential catalytic reactions. First, NADP is dephosphorylated to produce NAD, which catalytically activates a specific redox-cycle involving the enzymes alcohol dehydrogenase and diaphorase. During each turn of the cycle 1 molecule of a tetrazolium salt is reduced to an intensely coloured formazan. The method is capable of detecting as little as 0.01 amol alkaline phosphatase, and when applied to an immunoassay for TSH a sensitivity (zero + 2.5 standard deviations) of 0.0013 mIU/l was obtained.

Rabbit lung after inhalation of manganese chloride: a comparison with the effects of chlorides of nickel, cadmium, cobalt, and copper.

Rabbits were exposed to aerosols of MnCl2 (mass median aerodynamic diameter 1 micron) in metal concentrations of 1.1 and 3.9 mg/m3 for 4-6 weeks, 5 days/week, 6 h/day. The effects of alveolar type II cells, phospholipids, alveolar macrophages, and lung structure in general were compared with earlier reported effects of Ni2+, Cd2+, Cu2+, and Co2+. Except for a significant increase in the diameter of the alveolar macrophages after exposure to the higher Mn2+ concentration, no abnormalities were seen. The results of this and earlier studies indicate that these five metal ions have different, specific effects on the alveolar part of the lung.

Enzyme amplification can enhance both the speed and the sensitivity of immunoassays.

Enzyme immunoassays (EIA) are now used for the quantitation of a wide range of clinically important analytes and have, in many cases, replaced radioimmunoassays, though without improving on the sensitivity of the latter technique. We describe a general enzyme-amplification method which can be used to increase both the speed and the sensitivity of EIA. In this method, the enzyme label is used to catalyse the dephosphorylation of nicotinamide adenine dinucleotide phosphate (NADP+); the NAD+ so formed then catalytically activates an NAD+-specific redox cycle, yielding an intensely coloured formazan dye. The application of this new enzyme detection method has made possible an assay for human thyroid-stimulating hormone (TSH) with a sensitivity of 1 X 10(-5) IU/1 and a progesterone assay which takes only 15 min to complete.

An enzyme-amplified monoclonal immunoenzymometric assay for prostatic acid phosphatase.

An immunoassay for prostatic acid phosphatase is described in which a high degree of specificity for the prostatic isoenzyme, obtained by the use of monoclonal antibodies, is combined with great sensitivity, made possible by enzyme-amplified measurement of the combination of the isoenzyme with its antibody. The increase in sensitivity thus achieved is of the order of 170 times that of conventional methods of measurement. The advantages of the enzyme-amplified method have been shown to be particularly useful in detecting and monitoring small abnormalities of prostatic acid phosphatase levels in patients with prostatic cancer.

A new sensitive and fast peptide immunoassay based on enzyme amplification used in the determination of CGRP and the demonstration of its presence in the thyroid.

An enzyme amplified immunoassay for rCGRP based on cofactor cycling has been found to be clearly superior to a comparable radioimmunoassay employing the same antiserum in terms of sensitivity, speed and convenience. Correlation between the two methods was very good. With the enzyme amplified immunoassay we have been able to demonstrate the existence of rCGRP in thyroid extract.

A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry.

A method for greatly enhancing the sensitivity of assays employing enzyme labels is described which offers advantages in assays for a wide range of analytes. The principle of the new approach is that the enzyme label gives rise to a catalytic activator for a specific secondary detection system, the activity of which is measured and related back to the amount of label present and thus of the analyte it is being used to determine (C.H. Self, Eur. Pat. Appl. 80303478.4, 15.4.81 exclusively licenced to IQ (Bio) Ltd.). The general principle of enzyme amplification is illustrated by using alkaline phosphatase as the labelling enzyme and nicotinamide adenine dinucleotide phosphate (NADP) as its substrate. The nicotinamide adenine dinucleotide (NAD) formed catalytically activates a strictly NAD specific redox cycle which produces a coloured formazan as the end product. The measured absorbance is at least two orders of magnitude greater than that achieved by conventional methods. The application of this method to immunoassay is demonstrated by a sensitive, rapid and precise assay for human prostatic acid phosphatase (PAP). Some of the many other applications of this methodology are discussed.

Calcium signals and phospholipid methylation in eukaryotic cells.

Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.

Cholesterol in sarcoplasmic reticulum and the physiological significance of membrane fluidity.

Vesicles of sarcoplasmic reticulum from rabbit muscle can be loaded with cholesterol to at least 20 mol% with respect to endogenous sarcoplasmic-reticulum phospholipid without effect on the ATPase activity at 32 degrees C. This applies both to sarcoplasmic-reticulum vesicles in which the ATPase activity is stably coupled to Ca2+ accumulation, and to sarcoplasmic-reticulum vesicles in which the sarcoplasmic-reticulum ATPase is activated severalfold by fully uncoupling the enzyme from net Ca2+ accumulation. Since the incorporation of cholesterol causes a large decrease in fluidity of sarcoplasmic-reticulum phospholipid bilayer, these results for sarcoplasmic reticulum raise the more general question of whether bilayer fluidity is important in modulating the function of membrane proteins under physiological conditions as is widely assumed, or whether the function of membrane proteins may be effectively buffered under normal operating conditions against changes in bilayer fluidity due to extraneous agents.

The effect of bilayer thickness on the activity of (Na+ + K+)-ATPase.

The activities of purified (Na+ + K+)-ATPase supported by a series of phosphatidylcholines with monounsaturated (cis-9) fatty acyl chains (di(n : 1) phosphatidylcholine) varying in length from n = 12 to n = 23 were determined by the lipid titration technique. The ATPase activity at 20 degrees C decreased from 2.9 to 0.1 mumol/min per mg protein as n was decreased from 16 to 12 and decreased from 2.9 to 1.0 mumol/min per mg protein as n was increased from 20 to 23. In further experiments, the di(n : 1) phosphatidylcholine-ATPase complexes were treated with increasing proportions of n-decane, which has been shown previously to increase the thickness of black lipid membranes. n-Decane caused a large increase (greater than 20-fold) in activity of the short-chain complexes (n = 12,13); for n = 14--18, the ATPase activity first increased and subsequently decreased as the proportion of decane was increased, and for n = 20 or 23 decane caused a progressive decrease in activity with increasing concentration. These effects confirm qualitatively that a major factor determining the activity in each bilayer is its thickness. This behaviour closely parallels that of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum [1] and suggests that a major class of trans-membrane transport proteins may have a similar dependence on bilayer thickness.