Biomedical subjects
Publications and source records attributed to D J Sutor.
Common duct bile in patients with gallstones contains virtually no amylase activity. Therefore the raised levels of total CO2 concentrations found in common duct bile from patients with some calcium carbonate on the surface of their gallstones do not result from a reflux of pancreatic juice. A change in bile secretion is the likely explanation.
Eelven urine samples from normal subjects inhibited the formation of calcium phosphate. In all cases the activity of the urine was identical with that of an artificial urine having the same concentration of urea, creatinine, sodium, potassium, magnesium, calcium, ammonia, sulphate, chloride, inorganic phosphate, citrate, isocitrate and pyrophosphate, and the same pH.
Normal urine was examined for substances which inhibited the formation of calcium oxalate, using a separation scheme which involved ultrafiltration, alcohol precipitation, electrophoresis and paper chromatography. The major anionic inhibitors were found to be citric acid, isocitric acid and pyrophosphate. The inhibitory activity of urine and that of an artificial urine having the same concentration of urea, creatinine, sodium, potassiu, magnesium, calcium, ammonia, sulphate, chloride, inorganic phosphate, oxalate, citrate, isocitrate and pyrophosphate, and the same pH were identical. This confirmed that urine does not contain an unidentified potent substance which inhibits the formation of calcium oxalate.
When calcium and bicarbonate ions were mixed at room temperature (approximately 20 degrees C) to give concentrations of 4 mmol/1 and 21 mmol/1 respectively and the pH of the solution was kept at 8.3, vaterite, a form of calcium carbonate, was precipitated almost immediately as spheres of diameter 45 micron. The crystallisation of this material could be slowed down by adding to the crystallising medium small amounts of pyrophosphate or citrate which often inhibit crystal growth. High concentrations of sodium chloride (90 mmol/1) did not, however, affect the reaction. Very small amounts of gallbladder bile from patients with only cholesterol on the surface of their gallstones inhibited the crystallisation of calcium carbonate, and the size of the spheres was only 0.37 times those produced in water. The activity of the bile could be attributed to material with a molecular weight greater than 10 000. On the other hand, bile from patients having some calcium carbonate on the gallstone surface had less activity than comparable amounts of bile from patients with only cholesterol in this area. The active material may, therefore, play a part in preventing the deposition of calcium carbonate in gallstones.
An assay method has been developed for studying quantitatively the formation of calcium phosphate. The method will detect both substances which affect the solubility of calcium phosphate and substances which affect the crystal growth of the material. It is sensitive as or more sensitive than other methods described in the literature for detecting such compounds.
Normal urine has been examined for substances which inhibit formation of calcium phosphate. A separation scheme involving ultrafiltration, precipitation, electrophoresis and paper chromatography was devised to isolate these substances. Contrary to what has been suggested in the literature for many years, the urines examined did not contain a potent unidentified inhibitor. The major anionic inhibitors were citric acid, pyrophosphate and isocitric acid. These substances together with a small contribution from the cations appeared to account for most, if not all, of the inhibitory activity of urine.
The concentration of pyrophosphate in urine has been determined using the enzyme uridine-5'-diphosphoglucose pyrophosphorylase. The technique is relatively quick and easy to perform. Reproducibility of results was good, and results from recovery experiments were excellent. The mean concentration of pyrophosphate found in early morning urine samples from 14 normal adults was identical with that reported by other workers using an ion-exchange method to measure pyrophosphate. The pyrophosphate concentration was related approximately to the phosphate concentration.
The amount of D-isocitric acid in urine has been estimated using the enzyme isocitrate dehydrogenase. The technique is rapid and easy to perform. Reproducibility of results was good and results from recovery experiments were excellent. The concentration range for early morning urine samples from normal adults was 0.08-0.65 mmol/l with a mean of 0.29 mmol/l.
Measurement of total CO2 concentrations in bile from patients undergoing cholecystectomy because of gallstones has shown that the presence of calcium carbonate in the stones can be associated with a raised total CO2 concentration in the common duct bile. In bile from functioning and poorly-functioning gallbladders, total CO2 was nearly always related to pH irrespective of stone composition.
In gallbladder and common duct bile from patients undergoing cholecystectomy, usually because of gallstones, calcium was found to exist in at least 2 forms. Ultrafiltration showed some calcium was bound to substances with a molecular weight greater than 10 000, and the chief binding agent is likely to be the mixed micelle. Bound calcium was significantly less in common duct bile than in bile from functioning gallbladders, but the amount of ultrafiltrable calcium was the same. Furthermore, ultrafiltrable calcium in gallbladder bile from patients with cholesterol or some calcium carbonate in their gallstones was almost constant for a range of total calcium concentrations of 2.40--9.70 mmol/l. Comparison of ultrafiltrable and total calcium values for the different types of stone-formers showed that the deposition of calcium carbonate in gallstones was not related to any calcium measurement made. However, the presence of calcium phosphate and/or calcium bilirubinate in gallstones could be related to a significant increase in ultrafiltrable calcium in gallbladder bile.
The inorganic phosphorus concentration has been measured in fresh bile from patients undergoing cholecystectomy, usually because of cholelithiasis. The amount in common duct bile for patients with cholesterol on the stone surface was significantly higher than that for patients with some calcium carbonate in this area. For all patients with functioning gallbladders, inorganic phosphorus in gallbladder bile was always higher than that in the corresponding common duct bile. In bile from functioning gallbladders, inorganic phosphorus was linearly related to pH for cholesterol stone-formers, but values for calcium carbonate stone-formers were below the regression line and not related to pH. The results show that bile from calcium carbonate stone-formers is more dilute with respect to inorganic phosphorus.
1. The acid, neutral and "intermediate" salts of calcium bilirubinate have been synthesized. 2. All are crystalline, and the intermediate salt which has 1 1/2 molecules of bilirubin per calcium atom probably has a crystal structure containing molecules of both acid and neutral salts. 3. The acid or intermediate salts have been found in 14 out of 70 gallstones analysed recently by X-ray diffraction.
A series of 31 consecutive patients undergoing surgery for gallstone disease has been studied and the composition of the gallstones and bile from the gallbladder and common duct determined. As a result of the stone analysis by the X-ray powder diffraction method, the patients were classified according to whether their stones consisted of cholesterol, calcium salts or a mixture of the two. The mean composition of the common duct bile for the groups with cholesterol and mixed stones was just outside the micellar region of cholesterol solubility. The gallbladder bile from the cholesterol group of stone-formers was also supersaturated, but the gallblader bile from the group of mixed stone-formers was undersaturated with respect to cholesterol. None of the patients forming gallstones of calcium salts showed any abnormality in the cholesterol content of their bile.
The nucleation and crystal growth of calcium oxalate were studied under different conditions using a nephelometer to measure the turbidities of suspensions produced when dilute solutions of calcium chloride dihydrate and sodium oxalate were mixed. The amount of material produced was less when calcium was in excess of oxalate than vice versa. For equimolar amounts of the precipitating reagents the rate of formation of calcium oxalate was rapid when calcium and oxalate concentrations were 1-25 mmol/l. but slow when they were 0-5mmol/l. Sodium citrate and heparin inhibited nucleation and crystal growth and at certain concentrations prevented nucleation for the duration of the experiment. The method is simple and quick and requires only a small amount of material.
The pH of gallbladder and common duct bile from patients undergoing surgery for cholecystectomy has been measured. The bile was collected and kept anaerobically at 37 degrees C, and the pH measured at 37 degrees C, generally within 30-60 minutes of the bile being taken from the patient. Only data from patients having functioning or poorly-functioning gallbladders were included in the calculations. The pH of common duct bile was always greater than that of the corresponding gallbladder bile, and the data available suggest that it does not vary diurnally. The mean pH of gallbladder bile from patients undergoing their operation at different times during the day shows a diurnal variation, the bile becoming more acidic with time. Furthermore, there is also a diurnal variation in the activity of the acidifying process. Data for the patients suggest this is retarded by sleep, inactivity, or lying down.
When calcium and phosphate ions were mixed so that their final concentration was 4 mmol/1 and the pH was kept at 7-0, an amorphous precipitate immediately formed and this changed into crystalline material with an apatite-like structure after a period of time. The formation of either or both types of precipitate could be slowed down or prevented by adding to the crystallising medium trace amounts of pyrophosphate or citrate which are known inhibitors of the formation of calcium phosphate, or large quantities of sodium chloride which increased the ionic strength of the solution and hence the solubility of calcium phosphate, Both common duct and gallbladder bile from patients with gallstones composed of cholesterol and/or calcium carbonate had a very pronounced inhibitory action on the formation of these precipitates. Only very small amounts of bile were necessary to produce these effects, which therefore were not due to an increase in ionic strength. Ultrafiltration of bile showed that material with a molecular weight greater than 10 000 was mainly responsible for this activity. Because the inhibitor was present in both common duct and gallbladder bile, the liver is the likely source of origin. The possible identity of this material is examined. The powerful inhibitory effect of bile on the crystallisation of calcium phosphate is probably a contributory factor to the rare occurrence of the calcium phosphates, apatite and whitlockite, in gallstones.
A simple apparatus is described for the anaerobic ultrafiltration of a few millilitres of bile or other biological fluids. The principle employed enables viscous liquids which would normally block the membrane to be ultrafiltered successfully.
Out of a group of 979 patients with urinary calculi, 175 had passed their stone(s). Comparison of data for stones which had been passed with data for the whole group shows many significant differences. Calculi composed entirely of calcium oxalate are more likely to be expelled than any other composition variety. The chance of this happening is 1 in 3 and it is even higher when the person is under 50 years of age. The probability of calculi composed of calcium oxalate+calcium phosphate being expelled is 1 in 5 and this also increases for younger patients. Infection stones consisting of calcium phosphate+struvite have only 1 in 19 chance of being passed. Many of the stones passed (130) are under 0.10 g in weight. However, there is a similar relationshp between composition and weight among both the stones that were passed and the whole group. In both groups, pure oxalate stones are the lightest, infection stones are the heaviest and stones composed of calcium phosphate and calcium phosphate+calcium oxalate are of intermediate weight.