Role of hepatic anion-binding protein in bromsulphthalein conjugation.

Using gel filtration, the binding of both glutathione and Bromsulphthalein (BSP) to a liver-soluble protein was found to be identical. BSP-conjugating activity (glutathione S-aryltransferase) was present only in the fractions corresponding to the two protein-bound markers. Using a highly sensitive assay, with 3,4-dichloronitrobenzene, the pattern of glutathione S-aryltransferase activity was found to coincide with Y protein. This evidence suggests that Y protein, or ligandin, has a dual role in hepatic transport: a specific enzymic function in the conjugation of certain anions with glutathione in addition to a transport function in the intracellular binding of organic anions.

Synthesis and characterisation of an iodinated bile-salt derivative for photoaffinity labelling.

The synthesis and characterisation of a novel iodinated bile salt derivative, 125I-labelled 3 beta-azidocholylhistamine, is described. The derivative is handled by rat liver in a similar manner to taurocholate and binding to bovine serum albumin, a well-characterised bile acid-binding protein, is demonstrated. The suitability of the derivative for photoaffinity labelling is assessed.

Purification of bile acid-binding proteins from rat hepatic cytosol. Use of a photoaffinity label to detect novel Y' binders.

A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342-352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3 beta-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.

Studies in the rat on the hepatic subcellular distribution and billary excretion of lithocholic acid.

1. A compartmental model has been used to derive the in vivo subcellular distribution of lithocholic acid in rat liver. The model is based on the values of the partition coefficients for the distribution of lithocholic acid between subcellular fractions and buffer. It also permits calculation of the amount of lithocholic acid which is in free solution in cytosol. 2. The hypothesis that the rate of biliary excretion of a bile acid depends on the proportion in free solution was investigated by comparing the rates of biliary excretion of lithocholic acid and glycocholic acid. The rate for lithocholic acid was substantially less than for glycocholic acid while the percentages of each bile acid in free solution were 0.8% and 10%, respectively. 3. The validity of the model was supported by the observation that the amounts of lithocholic acid predicted to be present in the nuclear and cytosolic fractions were similar to the amounts found after intravenous injection of the bile acid.

A comparison of bile salt binding to lymph and plasma albumin in the rat.

The binding of bile salts to proteins in rat plasma and rat lymph has been investigated. Under the non-equilibrium conditions of gel chromatography no binding of glycochenodeoxycholate or glycocholate to any of the lymph proteins was observed. In contrast, plasma bound a proportion of both bile salts. When lymph was treated with charcoal, binding of glycochenodeoxycholate to a protein with a molecular weight identical to albumin occurred. Equilibrium binding studies showed that the binding of glycocholate to partially purified plasma albumin exhibited saturation kinetics with a dissociation constant of 2 x 10(-4) M. In contrast, the binding of glycocholate to lymph albumin was non-saturable. Potassium oleate, when added to plasma in a free fatty acid : albumin molar ratio of 3.8 : 1, almost completely inhibited the binding of chenodeoxycholate to plasma albumin. The endogenous free fatty acid : albumin ratios found in systemic plasma and lymph were 0.6 : 1 and 9.2 : 1, respectively. It is suggested that the high free fatty acid concentrations found in lymph inhibit the binding of bile salts to albumin.

A constant-volume ultrafiltration technique for the calculation of equilbrium binding data.

A constant-volume ultrafiltration technique is described, and details of its assessment presented. The retention characteristics of two membranes were evaluated using molecules of known molecular weight. The technique is rapid, precise, economical of material and yields equilibrium data. In these respects, it compares favourably with conventional techniques such as equilibrium dialysis.

Portal blood concentrations of conjugated cholic and chenodeoxycholic acids. Relationships to bile salt synthesis in liver cells.

1. Rats were maintained in a strictly controlled environment of 12 h illumination and 12 h darkness. At regular intervals during the light/dark cycle the portal blood conjugated cholic acid and conjugated chenodeoxycholic acid concentrations were measured. The bile salt concentrations exhibited similar diurnal rhythms, the highest concentrations occurring in the middle of the dark phase. 2. The concentrations of conjugated cholic and chenodeoxycholic acids in the portal blood of rats fed a diet containing the bile salt sequestrant, cholestyramine, were significantly lower than those found in rats given a control diet. 3. During total biliary drainage the portal blood concentrations of conjugated cholic and chenodeoxycholic acids fell to a minimum 6--8 h after the start of the experiment, whereas bile salt synthesis in hepatocytes isolated from the rats was not increased until the least 13 h after the commencement of total biliary drainage. 4. These results suggest that the concentrations of bile salts in the portal blood do not affect directly the diurnal fluctuation in rates of bile salt synthesis, as the response of synthesis to a change in portal blood bile salt concentrations is too slow. 5. When the rats were given a small supplement of cholesterol in the diet to suppress hepatic cholesterologenesis prior to being subjected to total biliary drainage, the changes in the portal blood concentrations of conjugated cholic and chenodeoxycholic acids and the synthesis of the two bile salts by isolated hepatocytes were similar to those found in rats given the control diet. 6. The rise in bile salt production seen during biliary drainage may not be dependent exclusively on a preceding increase in cholesterol synthesis.

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.

Increased collagen-linked fluorescence in skin of young patients with type I diabetes mellitus.

Our objective was to determine whether the fluorescence of skin collagen, which may reflect the accumulation of advanced glycosylation end products, is increased in young patients with type I (insulin-dependent) diabetes. Our study design was a cross-sectional case-control study in a referral-based diabetic clinic in an academic hospital. Study subjects comprised a convenience sample of 18 type I diabetic patients aged 17-30 yr and 8 age-matched healthy control subjects. The fluorescence of collagen was measured in skin biopsy material. Collagen-linked fluorescence (CLF) was increased in diabetic patients (mean 10.5 [range 5.8-15.8] U/mg) compared with control subjects (7.6 [5.6-10.1] U/mg, P less than 0.02). In diabetic patients, CLF was related to age (r = 0.581) and duration of diabetes (r = 0.697) but not concentration of glycosylated hemoglobin (r = 0.082). Partial correlation analysis demonstrated that duration of diabetes is the main factor determining the fluorescence of collagen in these patients. There was a relationship between CLF and presence of diabetic retinopathy after the data were adjusted for patient age and duration of diabetes (P = 0.023). Increased fluorescence of skin collagen can be detected in young type I diabetic patients and is primarily related to duration of diabetes.

Liver function in Edinburgh haemophiliacs: a five-year follow-up.

Liver function was assessed in 38 Edinburgh haemophiliacs. Results before the introduction of NHS intermediate factor VIII concentrate from 1974 onwards were compared with values in 1979. Measurements of serum bile salts in 16 patients as well as conventional liver function tests gave useful evidence of deranged liver function. Deterioration over the five-year follow-up period was seen only in patients on home treatment using large amounts of factor VIII concentrate, and there was no association between cryoprecipitate usage and derangement of liver function.

Precautions adopted in a clinical chemistry laboratory as a result of an outbreak of serum hepatitis affecting hospital staff.

This article describes the precautions which have been introduced into a clinical chemistry laboratory with a view to reducing the risk of infection of staff with the virus of serum hepatitis.

A rapid method for the measurement of plasma glucose concentration.

A modification of the manual glucose oxidase-gum guaiacum method of Shipton, B., Wood, P.J. and Marks, V. ((1975) Med. Lab. Tech. 32,33), for use with plasma, is described. The method enables rapid measurement of plasma or serum glucose concentration between 1 and 22 mumol/1 using 10 mul of sample and a single colour reagent. Experience of the method in routine use has shown that the coefficient of variation, beteen batch, is 2.1% and the time taken for analysis of 1 sample, standards and a quality of control sample is 12 min (including centrifugation of blood to obtain plasma).

An assessment of a reflectance meter system for measurement of plasma or blood glucose in the clinic or side ward.

The Reflomat System for rapid estimation of plasma or blood glucose concentration has been evaluated. The System gave a linear response throughout its analytical range and the recovery of glucose added to glucose-free plasma was 97-105%. Addition of sodium fluoride to plasma produced a 7-15% reduction in the estimated glucose concentration. Plasma glucose concentration estimated with the Reflomat agreed closely with results of a glucose oxidase and a hexokinase based method, and blood glucose concentration measured with the Reflomat agreed well with results of a glucose oxidase method.

Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid.

Investigations into the choice of immunogen, ligand, antiserum and assay conditions for the radioimmunoassay of conjugated cholic acid have been performed with a view to producing optimal assay conditions. Cholic acid-BSA was found to be the best immunogen to produce antibodies to conjugated cholic acid and the response was of an IgG type. Incorporating a spacer (hexanoic acid) between hapten and carrier protein resulted in a decrease in antiserum titre. Optimal conditions for the assay were found using [125I]histamine-glycocholic acid as ligand with a dilution of antiserum to produce 60% binding of ligand and a pH of 7.4. Using these assay conditions no serum effects were found; extraction of serum prior to assay was therefore unnecessary. The assay was sensitive enough to detect post-prandial increased in serum bile acid concentrations following a liquid test meal; no increase was observed throughout the same time period in a fasting control.

The preparation of 125I-labelled bile acid ligands for use in the radioimmunoassay of bile acids.

A general method for the preparation of 125I-labelled bile acid-histamine or 125I-labelled bile acid-tyramine conjugates is presented. The method is simple, quick and produces ligands in good yield (30%). The characteristics of a radioimmunoassay for conjugated chenodeoxycholic acid, based on an 125I-labelled ligand prepared by the method, are also described. The assay produced values for fasting serum concentrations of conjugated chenodeoxycholic acid that agree well with previous data.

The glyceryl [14C]tripalmitate breath test: a reassessment.

Several reports have been published commending the use of 14C-labelled triglyceride breath tests in the assessment of fat malabsorption. We report further studies using gyceryl [14C]tripalmitate. Corrections for age, weight or metabolic rate failed to improve the test's ability to discriminate between malabsorbers and control subjects. A correction for respiratory quotient improved the linear correlation observed between the breath test results and daily faecal fat excretion. The significance of these findings is discussed and a number of problems identified which, at present, are preventing the introduction of breath tests for fat malabsorption into routine clinical practice.

The effect of age on the intravenous sodium glycocholate test.

The clearance of an injected dose of sodium glycocholate has been studied in a group of 23 control subjects who had no evidence of liver disease. A significant correlation was found between bile salt clearance and age. No significant difference was found between the bile salt clearance of male and female subjects. Data derived from a simple compartmental model indicated that decreased clearance of bile salt with age was not specifically related to degenerative changes in hepatocellular integrity, hepatic uptake of bile salts or bile flow.

A recommended scheme for the evaluation of kits in the clinical laboratory.

A scheme for kit evaluation in the individual clinical chemistry laboratory is presented. Recommendations for assessing accuracy, precision, and stability are included along with a scheme for preparing for the evaluation and writing the final report.

Endogenous digoxin-like immunoreactive substance in patients undergoing coronary surgery. Preliminary report.

Perioperative digoxin concentrations were measured in 20 unselected adult patients undergoing coronary surgery. None of the patients were receiving treatment with digoxin. A digoxin-like immunoreactive substance was found in 16 patients postoperatively. This substance, if pharmacologically active, may have important clinical implications in the management of patients after open heart surgery.