Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins.

1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5'-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.


The role of intermediates in mitochondrial fatty acid oxidation.

1. Rat liver mitochondria oxidizing [16-14C]palmitoylcarnitine accumulate saturated long-chain thiester intermediates which may be detected by radio-g.1.c.2. Time-courses of intermediate accumulation display no product-precursor relationships and the end product, measured as [14C]citrate, is produced without a detectable initial lag. 3. A short pulse of [16-14C]palmitoylcarnitine followed by unlabelled palmitoylcarnitine showed that the observed intermediates(at least in the greater part)were not the direct precursors of [14C]citrate. 4. The quantity of saturated intermediates depended on the total accumulated flux of acyl units through the pathway provided that some mitochondrial CoA and unused substrate remained. 5. In the presence of rotenone and carnitine, 2-unsaturated, 3-unsaturated and 3-hydroxy intermediates were formed as well as saturated intermediates...


Antibodies to DNA in patients with rheumatoid arthritis and juvenile rheumatoid arthritis.

Antibodies to double-stranded DNA (DSDNA) were found in 18 patients with RA, in 5 patients with JRA, and in 5 patients with undiagnosed connective tissue disease. Five patients had clinical features consistent with both RA and SLE, 11 with only RA, and 5 with only JRA. Based on these observations, the presence of serum anti-DSDNA antibodies should not be used as a sole criterion in the diagnosis of SLE.


Development of antibodies to ribonucleoprotein following short-term therapy with procainamide.

An autoimmune response to certain nuclear antigens frequently develops in patients receiving prolonged therapy with procainamide. In order to define events involved in the initiation of this immune response, patients with myocardial infarction were studied early after starting procainamide and at later times. Polynucleotide antibodies and circulating polynucleotide antigens were sought by sensitive assay techniques in the sera of these patients. Very high titers of antiribonucleoprotein developed selectively in the majority of these patients after short-term therapy with procainamide. Such antibodies were infrequent in the long-term therapy group, most of whose members exhibited anti-single-strand DNA and were symptomatic with overt procainamide-induced lupus. Patients with acute myocardial infarction who did not receive procainamide did not develop anti-polynucleotide antibodies, but rather had high levels of free ribonucleoprotein antigen in their serum. Various interpretations of these data are discussed.


Ultrasonographic demonstration of popliteal cysts in rheumatoid arthritis. A noninvasive technique.

Ultrasound scanning techniques detected popliteal cysts in 14 of 24 knees affected with rheumatoid arthritis and associated with an anterior effusion. Serial scans demonstrated the persistence of cysts when effusion was not controlled by intraarticular steroid and lidocaine, and regression of cyst following control of effusion with anterior synovectomy. As the technique is noninvasive, painless, and reproducible, ultrasound scanning should be the technique of choice for the detection and assessment of popliteal cysts.


Genetic considerations of gout.

A great many genetic factors and genetic mechanisms are involved in the production of gouty arthritis. Genetic heterogeneity is to be expected in this disorder at all levels of expression. The genetic factors responsible for hyperuricemia, whether from gross overproduction of uric acid or from decreased renal excretion, can influence such practical matters as the selection of the proper therapy for control of the disease.


An overview of cellular and molecular mechanisms in cyrstal-induced inflammation.

In this account the authors have attempted to provide an introductory overview of the mechanisms of crystal-induced inflammation. More specific studies are given in other papers in this symposium. Like clinical gout itself, study of the sodium urate crystal has turned out to have a venerable heritage. Specific crystal identification has proved clinically useful.


Postsecretory reabsorption of urate in man.

These results are consistent with a model for renal tubular transport of urate in which there is reabsorption of both filtered and secreted urate. Urate secretion greatly exceeds total urate excretion, and most secreted urate is reabsorbed. At least a portion of urate reabsorption occurs at a site distal to or coextensive with the urate secretory site. There appear to be at least two distinct reabsorptive mechanisms for urate. The results of the flow rate and vasopressin studies are consistent with the hypothesis that urate reabsorption occurs in both the distal and the proximal tubule in man. The distal reabsorptive site appears to be quite small. It may be passive since it does not appear to be inhibited by uricosuric drugs. This reabsorptive site may account for less than 15% of total urate reabsorption. Both volume expansion and probenecid may inhibit urate absorption only in the proximal tubule. Thus reabsorption in the proximal tubule coud account for more than 90% of total urate reabsorption. Reabsorption at the postulated collecting duct reabsorptive site appears to be too small in magnitude to account for all reabsorptions of secreted urate. This could be explained if the reabsorptive site in the proximal tubule is coextensive with or distal to the secretory site. Alternatively, there might be two reabsorptive sites in the proximal tubule: a presecretory site accounting for the reabsorption of most filtered urate, and a site either coextensive or distal to the secretory site accounting for a major component of reabsorption of secreted urate. Finally urate reabsorption would also take place in the collecting duct, perhaps at a passive, flow-dependent site.


The influence of allopurinol on renal function in gout.

Our data demonstrate that adequate control of uric acid production and serum acid concentration in primary gout arrests further deterioration of renal function and in some instances permits subsequent improvement in renal hemodynamic function even when renal impairment is clearly established. In considering the nature of the lesions of urate nephropathy, our study suggests that control of serum uric acid with drug therapy before renal involvement is apparent might prevent or significantly delay the morbidity and mortality of this complication of primary gout.


The effect of hypercholesterolemia on aortic endothelium studied en face.

The aortic endothelium of rabbits fed a stock diet containing 1 or 2% cholesterol for intervals of 1 day to 21 weeks was compared with that of normal rabbits on a stock diet without cholesterol. The endothelium was examined en face using the Häutchen technique. The aortic endothelium of the experimental animals revealed an increase in stigmata and stomata, multinucleated giant cells and leukocytes particularly about sites of branching. These changes were also observed in the endothelium overlying lipid deposits but in addition, there was an increase in mitoses, an alteration in the orientation of endothelial cells and their nuclei, and a change in their argyrophilic staining properties. Closely related to the endothelium was an increasing number of foam cells which appeared to be derived from monocytic leukocytes.


Effects of palm-kernel oil and sunflower-seed oil on serum lipids and atherogenesis in alloxan-diabetic rabbits.

Groups of metabolically normal (controls) and alloxan-diabetic adult female rabbits were fed semi-synthetic diets containing 40 cal % palm-kernel oil (PKO) or sunflower-seed oil (SSO) for 54 weeks. In contrast to control rabbits fed PKO-diet, the alloxan-diabetic rabbits on this diet, developed no or only a negligible degree of atherosclerosis, although the serum levels of all lipid classes had increased in the diabetic rabbits above that of the controls during almost the whole experimental period. The diabetic rabbits and the controls fed SSO-diet were both free from any significant atherosclerotic involvement in spite of the fact that the SSO-diet appeared unable to suppress the very high levels of the various serum lipid classes induced by the diabetic state. On both diets, the diabetic rabbits showed a significantly higher cholesteryl linoleate/oleate ratio than the controls, which was caused by an increase in the cholesteryl linoleate level in the diabetics. No serious aorta atherosclerosis was found in rabbits with a cholesteryl linoleate/oleate ratio higher than 0.6, although no correlation was found between the atherosclerosis indices and these ratios at values lower than 0.6. Rabbits with cholesteryl linoleate/oleate ratios below 0.6 seemed to run a greater risk of developing atherosclerosis. It is suggested that insulin might be required for atherogenesis in addition to hyperlipemia and hypercholesterolemia.


Transfer of cholesterol in vitro between normal arterial smooth muscle tissue and serum lipoproteins of normo-lipidemic rabbits.

During incubation of normal arterial tissue with serum lipoproteins, net transfer of cholesterol was observed in the direction from the lipoproteins into the arterial tissue. Such transfer was only observed during incubations with single, isolated lipoprotein fractions. It was independent of the type of lipoprotein, VLDL, LDL or HDL, which was incubated with the arterial tissue. On the other hand, no net transfer of cholesterol was observed during incubations of arterial tissue with a combination of serum lipoproteins equivalent to that in native serum. Studies on the transfer of radioactive cholesterol suggested that cholesterol elimination from arterial tissue in vitro was not affected by the composition of the incubation medium. Therefore, it is suggested that cholesterol accumulation more easily is influenced by the serum lipoprotein composition, and that serum lipoprotein dysbalance may promote cholesterol accumulation in the tissue. This effect may be present even when the dysbalance involves a decrease of specific serum lipoprotein fractions.


The influence of atherogenic diet and "essential" phospholipids upon the contents of noradrenaline and dopamine in the brain of rats and their exploratory activity.

The influence of atherogenic diet and "essential" phospholipids (EPL) both on exploratory activity and catecholamine content in the brain of rats has been examined. The atherogenic diet caused a decrease of the animals' exploratory activity as well as a diminution of catecholamines. It was observed that these changes correlated. Prophylactic administration of EPL is capable of preventing such changes. Therapeutic administration of EPL increased exploratory activity and catecholamines in the atherogenic group which subsequently received the basic laboratory diet.


A study of different methods for the assay of lipoprotein lipase activity in human adipose tissue.

A method for the assay of lipoprotein lipase activity (LPLA) in heparin eluates of needle biopsies of adipose tissue is presented. A serum activated phosphatidestabilized emulsion of labelled triolein has been used as substrate. This method and a previously described method using heparin eluates as enzyme source and a commercial triglyceride emulsion, Ediol, as substrate, showed a high degree of correlation (correlation coefficient = 0.94) when parallel determinations were performed on biopsies from 16 subjects. Further, the Ediol method similarly correlated well with a method for LPLA assay, previously described by Nilsson-Ehle and Belfrage, on acetone-ether extracts of adipose tissue (correlation coefficient = 0.88; 19 subjects).


Long-term effects of colestipol (U-26,597 A) on plasma lipids in familial type II hyperbetalipoproteinaemia.

Results related to long term treatment with Colestipol (a new resin sequestering bile acids) in 23 subjects with familial hypercholesterolaemia, 12 with Type IIA, 8 with Type IIB and 3 homozygotes are reported. Patients were given 15 g/day active drug for a period of 12 months and a double dose (30 g/day) for a successive period of 4 months along with a low cholesterol, low saturated fat, polyunsaturated fat-rich diet. Mean cholesterol decrease was --42 +/- 18 mg/dl (P less than 0.05) after 12 months of 15 g/day Colestipol and --69 +/- 17 mg/dl (P less than 0.01) after the following 4 months of 30 g/day Colestipol. The difference between the two periods of treatment (15 g and 30 g/day was not statistically significant. A slight but not significant increase in triglyceride levels was observed. Serum uric acid showed a significant increase throughout the entire period of treatment. No malabsorption syndrome or signs of toxicity were seen. Most frequent side effects were constipation, nausea, and metheorism which, with the exception of 4 cases which were withdrawn from the study, were reported as being transitory and mild.


Rat heart lipoprotein lipase.

Rat heart lipoprotein lipase was highly purified by affinity chromatography using heparin-Sepharose 4B. When extracts of acetone powder were applied to columns, lipase activity was firmly bound to the gel matrix and was later eluted with 1.5 M NaCl. At this stage the eluted enzyme was purified 1500-fold. Disc gel electrophoresis yielded a single protein band corresponding with the enzyme activity. The apparent molecular weight was 60,000. The purified enzyme was highly unstable; however, its activity could be partially stabilized by glycerol or ethylene glycol. In studying the purified enzyme we observed: (i) a cofactor in serum was required for full enzymatic activity; ApoLp-Glu could be substituted for this cofactor; (ii) ApoLp-Ser was inhibitory to lipase activity; (iii) NaCl and protamine sulfate also markedly inhibited the lipase activity; (iv) heparin stimulated the enzymatic activity.


Abnormal post-heparin lipolytic activity in obesity. A preliminary note.

Published data have suggested that hypertriglyceridemia in obesity may result from the combination of hepatic overproduction and diminished removal of triglyceride-rich lipoproteins. Diminished catabolism might be expected if tissue lipoprotein lipase activity were decreased, a finding which has been reported in biopsies of adipose tissue from obese subjects. Abnormalities in heparin-released triglyceride lipase activity (PHLA) in obesity have not been reported, however. We have examined the possibility that methods for the measurement of PHLA might have failed to reveal such a defect because of the disproportionality between plasma volume and increasing body mass in obesity. Since it is usual to administer heparin on the basis of body weight, higher plasma heparin levels would be achieved in obese individuals. We performed standard PHLA assays in lean and obese volunteers. In the obese, heparin levels were consistently higher than in lean individuals although PHLA values were similar in both. Thus, PHLA in obesity appeared to be inappropriate for the heparin levels attained in plasma. Pharmacokinetic studies suggest that a decrease in PHLA available for release by heparin rather than heparin insensitivity underlies this phenomenon.


Relationship between the type of dietary fatty acid and arterial thrombosis tendency in rats.

The effect of dietary fatty acids on the formation and growth of intra-arterial occlusive thrombi in rats was investigated. It appeared that fats containing a large amount of saturated fatty acids promote arterial thrombus formation, whereas dietary linoleic acid has a specific anti-thrombotic effect. Oleic acid on its own does not seem to act as an anti-thrombotic substance. However, the replacement of thrombogenic fatty acids by oleic acid results in a decrease of the dietary thrombogenic potency. Moreover, the results indicated that the thrombogenicity of the saturated fatty acids increases with their chain length. As for the anti-thrombotic effect of cis fatty acids and their trans isomers, no obvious differences have been observed. Almost identical results were obtained when thrombogenicity was related to either absorption or to composition of the dietary fatty acids. Further research can therefore be restricted to this latter criterion. For the majority of the fats tested, thrombosis tendency and ADP-induced platelet aggregation were closely associated. It is therefore highly probable that platelet aggregation is involved in the mechanism by which dietary fats affect arterial thrombus formation.


The effect of renal hypertension on the regional deposition of cholesterol and phospholipid in the aorta of normally- and cholesterol-fed rabbits.

The effect of renal hypertension on dry defatted tissue mass and lipid accumulation in different segments of the aortic intima was studied in both normally-fed and cholesterol-fed rabbits. In normally-fed rabbits hypertension caused an increase in intimal dry weight in the aorta. The increase was greatest in the lower thoracic intimal segment but was not significant in the aortic arch. The increase in tissue mass was not influenced by the addition of cholesterol to the diet and no regression of the increased tissue mass occurred when a 4-week period of hypertension was followed by a 4-week period of normotension. Hypertension did not increase the intimal cholesterol or phospholipid concentrations in normally-fed rabbits, suggesting that an observed increase in lipid content represented the cellular component of the intimal hypertrophy. Hypertension in cholesterol-fed animals caused preferential lipid accumulation in the lower thoracic segment, an effect that was independent of the total intimal cholesterol level. Intimal cholesterol, cholesterol ester and phospholipid were all increased. When a 4-week period of normotension and cholesterol feeding was preceded by a 4-week period of hypertension with normal feeding the amount of cholesterol deposited did not exceed that of the normotensive control, suggesting either that hypertension increased intimal permeability to lipid only in the presence of hypercholesterolaemia, or that healing of damaged intima had occurred before hypercholesterolaemia was fully established.


Fatty acyl CoA synthetase activity in normal and atherosclerotic rabbit aortic tissue.

The activity of fatty acyl CoA synthetase and fatty acyl CoA:cholesterol acyltransferase was determined in microsomal fractions from normal and atherosclerotic rabbit aortic tissue. No change in fatty acyl CoA synthetase activity was observed as a result of cholesterol feeding in contrast to the several-fold increase in the activity of fatty acyl CoA:cholesterol acyltransferase seen in atherosclerotic tissue. Inhibition of both enzymes was observed when clofibrate, or the tetrahydronapthyl analog of this drug were added in vitro. The inhibitory effects were most pronounced on the fatty acyl CoA:cholesterol acyltransferase.


Coagulation studies as a prognostic index in acute liver failure.

A coagulation screen has been performed on 12 patients with acute liver failure. Six died and six recovered. All six fatal cases developed a haemorrhagic state with haemostatic failure. An attempt has been made to delineate the various mechanisms for the production of the coagulation defect. The significance of the different haematological parameters in assessing prognosis has been  assessed. The study emphasizes the importance of the synthetic ability of the liver in determining survival prospects. A good correlation between the factor-VII level, which is a guide to liver synthesis, and recovery has been shown. The value of a specific factor-VII assay in acute liver failure appears considerable. Where this assay cannot be performed the clot opacity fibrinogen technique provides a reasonable guide to the prognosis. The presence or absence of DIC was not a determinant factor in survival in this series.


Whole-body and organ Vo2 changes with enflurane, isoflurane, and halothane.

This study was designed to determine the effects of enflurane on canine whole-body and individual organ oxygen consumption (Vo2). Whole-body, myocardial, splanchnic, renal, and skeletal muscle Vo2 were determined at enflurane concentrations equivalent to those used in previous studies with halothane and isoflurane. With increasing enflurane concentrations, whole-body Vo2 decreased progressively. The major component of the decrease was a reduction in myocardial Vo2 resulting from a decrease in myocardial external work as a result of a decrease in cardiac output and arterial pressure. Other organs contributed to a lesser extent to the overall decrease in whole-body Vo2. In each respect the findings with enflurance were not significantly different from those with halothane and isoflurane. These findings did support to the view that anaesthetic agents are not general metabolic depressants and that observed changes in whole-body Vo2 reflect the summated changes in individual organ Vo2 occasioned by an anaesthetic-induced change in organ function and metabolic requirements.


Changes in plasma lipoprotein constituents during constant infusions of heparin.

Constant infusions of heparin of 4 to 6 hours' duration were used to estimate the transfer of very low density lipoprotein constituents to other plasma lipoproteins. Eleven subjects were studied, 3 with Type III and the remainder with either Type IV or V hyperlipoproteinemia. Whereas only about 5% of the triglyceride lost from VLDL was recovered in the other lipoproteins, 44% of the cholesterol was retained in the circulation, in equal amounts within low density (d 1.019-1.063) and intermediate density (d 1.006-1.019) lipoproteins. By contrast, there was no apparent loss of protein, more than half of that originally in VLDL being recovered in high density lipoprotein. In subjects with Type III hyperlipoproteinemia, lipid and protein was lost from the intermediate density lipoprotein as well as from VLDL. In subjects with marked hypertriglyceridemia, cholesterol became redistributed from larger to smaller VLDL. The esterification of plasma cholesterol as measured in vitro, was apparently suppressed during the early phase of the heparin infusions, but tended to recover later.


The combined effect of halothane-induced hypotension and hypocapnia on canine myocardial blood-flow and oxygen consumption.

In eleven intact mechanically ventilated dogs anaesthetized with pentobarbitone, mean arterial pressure was reduced to 55% of the control value by ventilating with 1-1.5% halothane. At normocapnia this resulted in decreases in myocardial blood-flow and oxygen consumption to 47% and 55% of their respective control values and an increase in myocardial vascular resistance to 123% of control value. When hypocapnia (mean PaCO2=26 mm Hg) was superimposed on hypotension, myocardial blood-flow was further decreased to 38% of control value whereas oxygen consumption did not alter significantly. When halothane was discontinued, allowing recovery from hypotension, the rate of recovery of myocardial blood-flow and cardiac output was much slower than that of arterial pressure.


Effect of volatile anaesthetics on cell survival as measured by colony forming ability.

Chinese hamster fibroblast cells in stirred suspension cultures were exposed to volatile anaesthetic agents for 3 hr or for 24 hr. Cell viability was measured as colony forming ability and was found to be reduced, in a dose related manner, by halothane, chloroform, methoxyflurane, trichloroetheylene and diethyl ether.


Aortic washout of 125I compounds in atherosclerotic and normal rabbits.

An in vivo rabbit model is described for the study of aortic washout of 125I tagged compounds. Washout is compartmental and in sequence from the lumen, aortic wall, and periaortic fat. Toluene-azo-naphthol (TAN) affixed chiefly to periaortic fat. A double isotope method with TAN plus a cholesterophilic substance is suggested for the assay of atherosclerosis in situ.


Crystals in atherosclerotic lesions: real or artefact?

Atherosclerotic lesions were obtained from man during surgery and from cholesterol-fed rabbits. They were maintained at about 37 degrees C during handling. Smears were prepared on glass slides and these were examined microscopically at 37 degrees C. Solid rhomboidal or thick needle-like crystals were present at 37 degrees C but increased in numbers or in size or both on cooling. Staining studies and measurement of melting point (133-153 degrees C) suggested that such crystals are composed largely of free cholesterol or a related sterol. Liquid crystals exhibiting conic focal (Maltese cross) anisotropism were present at 37 degrees C and did not appreciably increase in either size or numbers on cooling. Staining studies and their resistance to digitonin suggested that these Maltese cross crystals are largely esterified cholesterol. Thin needle-like crystals arranged like feathers or in rosettes were seen in smears of adipose tissue and were attributed to triglycerides.


Effect of althesin (CT-1341) on thyroid-adrenal function in man.

The effects of althesin on adrenocortical and thyroid function were investigated by measuring plasma concentrations of cortisol, thyroxine (T4) and free thyroxine index (FTI) in 29 surgical patients. Althesin did not change the plasma cortisol concentrations from the preanaesthetic control value; however, the concentrations increased significantly after the start of surgery and thereafter. Althesin anaesthesia per se decreased slightly, but significantly, plasma T4 concentration, but did not influence the FTI value. The surgical operation increased the FTI value slightly but significantly. Our data would suggest that Althesin anaesthesia can be used in patients who have abnormal adrenocortical or thyroid activity.


Dose relationships of phase II, tachyphylaxis and train-of-four fade in suxamethonium-induced dual neuromuscular block in man.

In man under halothane-nitrous oxide-oxygen anaesthesia, suxamethonium-induced neuromuscular block can be divided into two phases using the train-of-four ratio: the first phase of minimal train-of-four fade and the second phase of marked train-of-four fade. The second phase is characterized by a low train-of-four ratio, 0.3 or less, and resembles tubocurarine-block) in many details. It corresponds to Phase II (dual block in the literature. Contrary to the prevailing belief, Phase II block has a rather abrupt onset. In this study of seven patients, Phase I covered up to a total of 1--3 mg/kg of suxamethonium, while 3--5 mg/kg established Phase II block, with a transitional zone of only 2 mg/kg. Establishment of Phase II also coincided with onset of unequivocal tachyphylaxis. The possibility of a causal relationship between tachyphylaxis and Phase II block is suggested.


Investigations of fluorescent peptides and lipofuscins of human intervertebral disc relating to atherosclerosis.

(1) Annulus fibrosus and nucleus pulposus of human intervertebral discs at different degrees of atherosclerosis were disintegrated by elastase. (2) The material disintegrated by elastase -- called elastolysate -- could be separated into hydrophobic (apolar) and hydrophilic (polar) peptides. Parallel with the degree of atherosclerosis the amount of hydrophobic peptides increased, whereas that of the hydrophilic peptides decreased. (3) In annulus fibrosus and nucleus pulposus two kinds of fluorescence maxima were measured. The one, A:F 350:405, is known as fluorescence maxima of elastin- and collagen-peptides. The other, A:F 410:470, is related to a similar substance called atherofluorescent component (AFC), which has been isolated before from the plaques of atherosclerotic aorta. This substance accumulates mainly in nucleus pulposus and resembles lipofuscin-like bodies. (4) These bodies show a positive reaction with thiobarbituric acid, giving a red coloration characteristic for malondialdehyde. In nucleus pulposus the amount of lipofuscin-like substances is much greater than in annulus fibrosus. (5) The hydrophilic peptides, although they show the same fluorescence maxima as the hydrophobic peptides, do not give any reaction with thiobarbituric acid. It is supposed that in these cases the cross-linked protein contains instead of malondialdehyde other reactive aldehydes.


Mean skin temperature during anaesthesia: an assessment of formulae in the supine surgical patient.

The mean skin temperature was calculated from measurements made in 22 patients under general anaesthesia. The results from 11 different formulae were compared with a system involving 15 temperature sites on the skin, each given a weighting factor in proportion to the fraction of total surface area which it represented. If accuracy is required, the mean skin temperature should be measured in surgical patients using a formula which involves at least 10 skin sites. Incorrect estimation of stored body heat results from an erroneous value for the mean skin temperature, and this occurred more frequently when the number of skin sites was reduced.


Growth hormone response to insulin and to arginine in patients with familial hypercholesterolaemia.

Human growth hormone (HGH) response to i.v. insulin (0.1 U/kg body weight) and arginine infusion (25 g of L-arginine for 30 min) was studied in 9 patients (5 males and 4 females) with primary familial hypercholesterolaemia and belonging to 4 families. Mean age was 28 +/- 2 years (range 18-36) and body weight was less than 105% of ideal body weight. Glucose tolerance and insulin response to oral glucose were normal in all patients. HGH release after insulin and after arginine was slightly increased as compared to 21 normal controls, but the differences were not significant. Insulin and glucagon response to arginine in these patients was within the normal range. Plasma glucose and free fatty acids were normal after both insulin and arginine. Moreover, no significant correlation was found between fasting cholesterol and HGH peaks after insulin and after arginine, nor between cholesterol and insulin and glucagon responses. Despite marked hyperlipidaemia, HGH-deficient patients examined by other authors never present signs of atherosclerotic disease. Our data suggest that HGH, in the presence of elevated cholesterol levels, might play an important role in the development of atherosclerotic lesions.


Effects of althesin anaesthesia and surgery on carbohydrate and fat metabolism in man.

The effects of Althesin anaesthesia alone, and associated with surgery, on carbohydrate and fet metabolism were studied in 36 patients by determining the plasma concentrations of cortisol, human growth hormone, insulin, free fatty acids, and blood sugar. The differences between the mean blood sugar and plasma cortisol concentrations before induction and during anaesthesia alone were not statistically significant. Blood sugar concentrations increased slightly during minor surgery and increased significantly during body surface and abdominal surgery. The plasma cortisol concentrations increased slightly during minor and body surface surgery but increased significantly during abdominal surgery. Plasma insulin and human growth hormone concentrations did not change appreciably during anesthesia and surgery. During Althesin anaesthesia alone and during minor surgery, a significant increase in plasma FFA concentration was observed at 15 min. The hyperglycaemia response in diabetic patients did not differ significantly from that in nondiabetic subjects. The clinical significance of the results is discussed.


Induction and recovery with enflurane and halothane in paediatric anaesthesia.

One hundred and four infants and children were included in a comparative study of anesthesia with either enflurane or halothane. Induction and recovery were significantly shorter with enflurane than with halothane. There was little difference between the drugs in respect of their postoperative sequelae.


Minimum blood concentration--a universal concept of anaesthetic potency.

Although brain concentration of anaesthetic agents is attractive as a method of expressing anaesthetic potency, both theoretical and practical considerations limit the usefulness of this index. Therefore, the MAC concept has been utilized to develop another expression of potency: the minimum blood concentration. This takes into account the blood/gas solubility coefficient, and the fact that at equilibrium the amount of anaesthetic agent present in brain is reflected in the blood concentration, and it is applicable to both parenteral and to inhalation anaesthetic agents.


Hexafluorenium-suxamethonium interaction in patients with normal versus atypical cholinesterase.

The effect of hexafluorenium 0.3 mg/kg on the neuromuscular block of suxamethonium 0.1 mg/kg in a child with homozygote atypical plasma cholinesterase activity and in one of his normal brothers has been compared. In the normal child, hexafluorenium produced a marked potentiation of suxamethonium block, while partially antagonizing the block in the atypical homozygote child. In both situations, the hexafluorenium-suxamethonium block appeared to be antidepolarizing in nature as indicated by tetanic fade and post-tetanic facilitation. It is concluded that, in man, hexafluorenium can modify the action of suxamethonium by two different mechanisms. In patients with normal plasma cholinesterase activity, the anti-esterase effect predominates and results in marked potentiation of suxamethonium block, while in atypical homozygotes a direct antidepolarizing action at the neuromuscular junction may result in a diminished response.


A potentially serious complication with Swan-Ganz catheter placement by the percutaneous internal jugular route.

Percutaneous insertion of a Swan-Ganz catheter through the internal jugular vein is presumed to have resulted in lodgement of the catheter tip in the wall of the internal carotid artery.


Postoperative hypoglycaemic coma associated with chlorpropamide.

A 72-year-old male being treated with chlorpropamide for diabetes mellitus had an emergency operation for a perforated gastric ulcer. Hypoglycaemic coma occurred after the operation.


Potassium supplements in patients treated with corticosteroids.

1. Preliminary studies are reported on 8 patients with lung disease given prednisone both with and without potassium supplements. 2. There were no abnormalities of plasma potassium but there was a relationship between the dose of prednisone and the urinary excretion of potassium whilst off supplements: higher doses were associated with increased potassium excretion. 3. Patients who had been on treatment for a short period retained more of their supplements than did those who had been on treatment for several years. 4. It is suggested that with prolonged treatment control of potassium homeostasis may be altered, and that more detailed metabolic studies should be carried out.


Lung function in childhood. III. Measurement of airflow resistance in healthy children.

Indices of airflow resistance have been obtained in a group of 248 healthy London schoolchildren from recordings of peak expiratory flow rate (PEFR), forced expiratory volume in 0-75 seconds (FEV 0-75) and maximum mid-expiratory flow (MMEF) and two more direct methods, airways resistance (Raw) measured plethysmographyically and total respiratory resistance (PT) by the forced oscillation technique. The normal data are presented.


Treatment of advanced bronchogenic carcinoma with adriamycin and 5-fluorouracil.

Thirty patients with an advanced bronchogenic carcinoma were treated with a combination of adriamycin and 5-fluorouracil; in eight the size of the tumour or its metastases was reduced by over 50%, and eight further patients experienced useful relief of symptoms. This drug combination is useful in the magagement of lung cancer.


Sputum and serum levels of amoxycillin in chronic bronchial infections.

A study of 11 patients suffering from acute exacerbations of chronic bronchial infection, all with purulent (Mp+++) sputum, showed that good penetration of amoxycillin into such sputum occurs. The levels of amoxycillin in sputum doubled proportionally with the oral dose, at least up to dosages of 2 g. High dosages or oral amoxycillin may therefore, be advantageous in the treatment of chronic bronchial infection. In a study of 30 chronic bronchitic patients, whose sputum varied in purulence from mucoid to purulent (Mp+++) the concentration of amoxycillin in sputum containing approximately 50% pus (Mp++) was significantly higher than that in any other degree of purulence. These results indicate that amoxycillin penetrates best into sputum at an intermediate degree of purulence.


Pigmented basal cell epithelioma. Light and electron microscopic studies on tumours and cell cultures.

Light and electron microscopic studies have been carried out on three pigmented basal cell epitheliomas and on cell cultures of two of these tumours, to determine the formation and transfer of pigment and the fine structure of the constituent cells. Scattered in the epithelial tumour buds were many functional melanocytes that contained melanosomes in various stages of development and whose morphology was essentially normal. Many melanophages were present in the dermal stroma, but few transferred melanosomes were to be found in the epithelial tumour cells. Tissue culture studies revealed that melanocytes were present among the proliferating islands of epithelial cells and these contained many melanosomes. However, few melanosomes were found in the epithelial cells. This study indicated a block in the transfer of pigment from the melanocyte to the basal epithelioma cells. Several of the cultured epithelial cells were found to contain dense bundles of filaments resembling amyloid. It is likely that they are abnormal tonofilaments resulting from the differentiation and keratinization of basal cells in vitro.


Pigmentary demarcation lines. Comparison of Negroes with Japanese.

The natural pigmentary demarcation boundaries of the skin can be classified as: Group A--lines along the upper limb with variable trans-pectoral extension; Group B--lines along the lower limb; Group C--paired lines in median or paramedian course on the chest with midline abdominal extension; Group D--posteromedian demarcation; and Group E--bilaterally symmetrical, obliquely oriented hypopigmented macules on the chest. Groups A and C are the most distinct and occur in both Negroes and Japanese.


Median rhomboid glossitis. Candidiasis and not a developmental anomaly.

Infection by Candida Albicans was found in all biopsies taken from ten patients presenting with midline lesions of the tongue which clinically presented as median rhomboid glossitis.


Dermo--epidermal adhesion and its effect on epidermal structure in the mouse.

Evidence is presented that dermo--epidermal adhesion is low in mouse ear. Consequently the pressure caused by mitotic activity in the basal epidermal layer is readily relieved by cell extrusion distally and the number of basal cells per unit skin area remains low. The rule then is that the number of distal cells per unit skin area must be equally low, and the epidermis is therefore thin. Conversely, dermo--epidermal adhesion is high in mouse sole-of-foot epidermis, and the mitotic pressure leads through cell crowding to a columnar basal epithelium. Furthermore, in the footpad centre, where adhesion is evidently highest, the baseline folds. With basal cell crowding and baseline folding there are more basal cells, and therefore more distal cells, per unit skin area. The epidermis is thick. Epidermal cells are regarded as equipotential. The different epidermal characteristics of the different body regions are determined by the combined actions of the local mitotic rate and the local strength of the dermal grip.


An improved method of assessing topical corticosteroid activity.

Topical application of ointment bases causes varying degrees of epidermal thickening in guinea-pigs. This is reproducible and can be accurately measured. Suppression of these changes was produced by addition of beta-methasone 17-valerate, fluocinolone acetonide, fluocinonide and hydrocortisone acetate. The inhibitory effect of these steroids was related to the type of corticosteriod, its concentration and the ointment base. The effect was still evident at extremely high dilutions of the steroids and could be measured at concentrations as low as 5 X 10(-5) % of fluocinonide in FAPG. This technique has the merits of being reproducible, sensitive and accurate. It should find a place among existing methods in assaying the efficacy of topical corticosteroids and in aiding in the selection of bases most suitable for formulation of these preparations.


Lichen planus and ulcerative colitis.

Three patients with ulcerative colitis and lichen planus are reported and similar cases in the literature discussed. Similarities in the pathology of the two conditions are outlined.


The development of radioimmunoassays for fibrinogen degradation products: fragments D and E.

Fibrinogen degradation products, fragment D (FgD) and fragment E (FgE) have been measured in human serum by specific radioimmunoassays. In addition, the appearance of a neoantigenic determinant on FgD, revealed when fibrinogen is degraded by plasmin has been utilized to develop a specific radioimmunoassay for FgD in plasma (FgDneo). The reagents and conditions used in each assay are described in detail. The mean specific activity was 144 muCi/mug for 125I-labelled FgE and 82 muCi/mug for 125I-labelled FgD. Separation of antibody bound and free antigen was achieved using second antibody. The detection limits of the FgE, FgD and FgDneo assays were 0.8, 1.0 and 6.2 ng/ml respectively. The specificity of each assay with respect to fibrinogen and its degradation fragments has been assessed. Fibrinogen and fragment X cross-reacted markedly in both the FgE and FgD assays, whereas the cross-reaction of fibrinogen was abolished in the FgDneo assay, while the cross-reaction of fragment X was 10%, indicating gradual emergence of the neoantigenic site during digestion of fibriogen. The sensitivity, precision, and specificity of the radioimmunoassay systems described have major advantages over the existing procedures for the measurement of fibrinogen degradation products.


The subcellular localization of the erythrostimulant factors present in the kidney and spleen of the rabbit.

The subcellular localization of erythrostimulant factors contained in the kidney and spleen of the rabbit have been studied. This was done by subjecting homogenates to fractionation by centrifugation and assessing the erythrostimulant capacity of the hypotonic extracts prepared from the various fractions thus obtained.  The results of these studies show that the erythrostimulant factors are present in the light mitochondrial and microsomal fractions of the normal kidney. In conditions of anaemia they are to be found not only in the above-mentioned fractions but also in the mitochondrial fraction. By contrast, in the spleen such factors are present only in the mitochondrial fraction. It is likely that the different localization of the erythrostimulant factors in the kidney and in the spleen of the rabbit may be closely connected with the different functions performed by these organs in the humoral regulation of erythropoiesis.


Globin chain synthesis in the greek type (A gamma) of hereditary persisitence of fetal haemoglobin.

Globin chain synthesis was studied in a family with both the Greek (Agamma) type of hereditary persistence of fetal haemoglobin and beta thalassaemia. The ratio of alpha/(gamma+beta+delta) chain synthesis in the hereditary persistence of fetal haemoglobin (HPFH) heterozygotes was 0.97 while in the HPFH/beta-thalassaemia heterozygote it was 2.14. However, calculation of the amounts of haemoglobin synthesized per cell suggests that in the HPFH/beta-thalassaemia heterozygote, the beta- and Agamma-chain genes in cis to the HPFH determinant are unable to compensate for the deficiency of chains imposed by the beta-thalassaemia gene in trans and that the increased synthesis of Hb F is directed by the gamma-chain genes located on the beta-thalassaemia chromosome. The data suggest that synthesis of beta and Agamma chains in the Greek HPFH is fixed at a 'preset' level and indicate that the defect might be due to an abnormality in the rate of transcription of the closely linked beta-, delta- and gamma-chain genes.


Four new pyruvate kinase (PK) variants and a classical PK deficiency.

Four new red-cell pyruvate kinase (PK) variants are presented along with one case of so-called classical type PK deficiency. PK 'Tokyo II' had a low activity, Km (PEP) and Vmax, but a normal urea stability and only slight deviation from normal in neutralization tests by antiserum. It had a normal nucleotide specificity, abnormal electrophoretic mobility (fast moving) and the variant was associated with a mild hemolytic anaemia. PK 'Maebashi' had a low activity, high Km (PEP), low Vmax, urea instability, decreased reactivity to antiserum, normal electrophoretic mobility, normal nucleotide specificity and was associated with a moderate haemolytic anaemia. PK 'Tsukiji' had low activity, high Km (PEP), markedly high Vmax, urea instability, decreased reactivity to antiserum, abnormal electrophoretic mobility (fast moving) and grossly abnormal nucleotide specificity especially abnormal behaviour to ADP. The haemolytic process in this case was moderate to severe. PK 'Ube' was electrophoretically abnormal (fast moving) but otherwise had normal characteristics and the propositus was healthy and not anaemic. PK 'Ube' was found by electrophoretic screening for genetic PK polymorphism. In the classical type PK deficiency, the usual red-cell PK (PK-R1 and PK-R2) was not demonstrable by electrophoresis but instead M2-type PK was present, presumably by compensatory process. Kinetic studies confirmed that the patient's red-cell PK consisted of M2-type PK. This patient had a severe haemolytic anaemia.


Chamber centrifugation: a harvesting technique for estimation of the growth of human haemopoietic cells in diffusion chambers.

A harvesting technique which gives information about the spatial relationships between cells grown in diffusion chambers in mice has been designed. The principle is that cells are centrifuged directly from the chamber clot onto a slide during pronase treatment. The technique has been applied to normal human bone marrow cells grown in diffusion chambers. The slides show that the cells are situated in clusters. A limiting dilution assay of progenitor cells based on the presence of granulopoietic clusters in the chambers demonstrates the use of the method for quantitation of progenitor cells.


Lymphocyte transformation in patients with paraproteinaemia.

The transformation of lymphocytes in cultures with phytohaemagglutinin (PHA) or with pokeweed mitogen (PWM) was carried out during treatment on 28 patients with paraproteinaemia and on 20 control subjects. 78.5% of the peripheral blood lymphocyte short-term cultures from the treated patients showed a subnormal PHA-induced but high PWM-induced lymphocyte transformation. The other 21.5% of the treated patients had a normal pattern of lymphocyte transformation. Eight of 12 untreated patients with multiple myelomatosis and four of five untreated patients with benign paraproteinaemia also had a subnormal lymphocyte transformation to PHA but a high response to PWM. Serial lymphocyte transformation studies in eight of 18 myeloma patients during chemotherapy revealed that the initial subnormal lymphocyte transformation to PHA subsequently became higher than that to PWM. 82% of the bone marrow cultures from the patients showed a higher PWM-induced than PHA-induced lymphocyte response, also suggesting a B cell type of disease. In both dose response and time dependent investigations peak transformation of peripheral blood lymphocytes did not always coincide.


The effect of various cytotoxic agents on bone marrow progenitor cells as measured by diffusion chamber assays.

Two diffusion chamber assays, termed the 'Full" chamber technique and the 'Empty' chamber technique, have been used to determine the effect of various doses of cyclophosphamide, vinblastine and busulphan on the population of diffusion chamber progenitor cells (DCPC). The diffusion chamber dose-response curves were compared to the progenitor cell survivals estimated by the spleen colony technique. The in vitro agar colony assay was also performed on the busulphan-treated marrow. The diffusion chamber and spleen colony techniques estimated similar survivals after cyclophosphamide and vinblastine treatment. However, with busulphan, the chamber, spleen colony and agar colony methods estimated different survivals. The indication is that the diffusion chamber techniques assay the same population of cells as the spleen colony technique, and that busulphan alters the capacity of surviving DCPC to generate granulocytes and macrophages.


Kinetic studies of 51Cr and DF32P labelled granulocytes.

In vivo kinetic studies of granulocytes labelled in vitro with 51Cr and DF32P were carried out in nine haematologically normal subjects by isolation of the cells in the blood samples by the Ficoll-Isopaque flotation method. 51Cr and 32P specific activity of blood samples made of 93-98% granulocytes was studied. Distribution between marginated and circulating granulocyte pools was identical for both labelled cells and the marginated pool was similar to the circulating pool, except that it was lower in one subject who had a previous splenectomy. The half-disappearance time (T 1/2) was 16.1+/-2.2 h for 51Cr-labelled and 5.4+/-2.1 hr for DF32P-labelled granulocytes. In one case of a normal subject who previously received multiple transfusion homologous 51Cr-labelled granulocytes had a T 1/2 of less than 1 h.


The use of an image analysing computer for the quantitation of red cell morphological characteristics.

Measurements of integrated optical density and projected area of red cells have been made on Romanowsky-stained blood films from 25 subjects using an image analysing computer. Close correlation was observed between mean cell projected area and MCV and between mean cell integrated optical density and MCH. Mean values for the quotient of cell integrated density/cell projected area (mean cell colour density) correlated with MCHC. Varieties of cell area and colour density correlated subjectively with anisocytosis and anisochromasia, and histograms of these parameters demonstrated dual populations. The clinical application of these findings is discussed.


Measurement of heparin in patients receiving subcutaneous heparin therapy.

Heparin assays based on four different tests of clotting function have been compared with a heparin assay utilizing the potentiating effect of heparin on anti-factor Xa in a group of patients receiving subcutaneous heparin therapy during late pregnancy. It is considered that the anti-factor Xa method is a better indication of the antithrombotic effect of heparin. In the presence of high levels of clotting factors, heparin assays based on tests of clotting function can be misleading.


A comparison between radioimmunoassay and other immunological techniques for the measurement of fibrinogen/fibrin degradation products in serum.

Fibrinogen/fibrin degradation products (FDP) have been measured in serum by radioimmunoassay for fragment E (FgE), one of the terminal fragments in plasmic digestion of fibrinogen, and the results compared with those determined by both a tanned red cell haemagglutination inhibiton immunoassay (TRCHII) and a latex particle agglutination inhibition immunoassay. The detection limit of the FgE assay was 0.8 ng/ml, that of TRCHII was 625 ng/ml and the latex particle agglunitaion inhibition immunoassay was 10 mug/ml. All the samples measured had detectable levels of FDP with the FgE assay, whereas only 88% were measurable with the TRCHII and 2% by the latex particle agglutination inhibition immunoassay. A comparison of those samples giving a positive result with both the TRCHII and FgE assay showed overall agreement between the results of the two types of assay, but there was considerable scatter of FgE levels at each point of the TRCHII. The major advantages of the radioimmunoassay system are greater sensitivity, specificity and reproducibility.


Coagulation changes during the steady state in homozygous sickle-cell disease in Jamaica.

Coagulation studies were carried out in 117 Jamaicans with homozygous sickle-cell disease in the steady state, and 40 local controls. The patients had significantly higher factor-VIII levels, higher platelet counts, lower factor-V and plasminogen levels, shorter thrombin times and higher serum fibrinogen degradation products (FDP) than the control group. The low factor-V and plasminogen levels, and high FDP levels, might be explained by activation of the coagulation system and continuous clot lysis even in the absence of painful crisis. The high factor-VIII levels and short thrombin times found in these patients could not be explained.


Erythraemia due to haemoglobin San Diego.

A 63-year-old man with erythraemia was found to have a high affinity haemoglobin which was subsequently identified as Hb San Diego beta 109 (G11) Val leads to Met, recently described in a Filipino family (Nute et al, 1974). The oxygen affinity of the haemolysates was increased approximately two-fold at pH values between 6.04 and 7.90. The n value was nearly normal (2.5-2.9) in the upper range of saturation (70-95% oxygenation) but was somewhat diminished (1.9-2.1) below this range. In vitro biosynthesis of globin by reticulocytes showed that there was balanced synthesis of alpha and beta chains (alpha:beta ratio = 1).


The oxygen affinity of haemoglobin E.

Oxygen dissociation studies were carried out on haemoglobin E (Hb E) at both high and low haemoglobin concentrations. Oxygen affinities of fresh red cells from three people homozygous for Hb E and from one with Hb E-beta thalassaemia (Hb-E trait/beta-thal trait) were low in three out of four patients studied, while the oxygen affinity of red cells from an individual with Hb-E was normal 2,3-DPG concentration in the fresh cells from the people with homozygous Hb E or Hb-E trait/beta-thal trait which showed low oxygen affinities were elevated sufficiently to account for the shifts observed. When the cells from two of these people with homozygous Hb E were depleted of 2,3-DPG. their oxygen affinities became the same as that of similarly treated normal cells. Pure 'stripped' Hb E in dilute solution behaved identically to Hb A in respect of P50, Bohr shift, haem-haem interaction, and interaction with inorganic phosphate or 2,3-DPG. Hb E, therefore, has the same oxypgen dissociation properties as Hb A both in dilute solution and in the red cell. The low oxygen affinities found in the fresh cells and in whole blood are caused by high 2,3-DPG concentrations within the cell.


The oxygen affinity of haemoglobin Hammersmith.

Oxygen dissociation studies were carried out on red cells and lysates from a patient heterozygous for Hb Hammersmith. The oxygen affinity of the cells at pH 7.1 was decreased, partly by an increased cellular concentration of 2,3-DPG and more importantly by an intrinsic low affinity of Hb Hammersmity. Haem-haem interactions were reduced and an abnormal Bohr effect (pH 7.1-7.4) was found in the cells which would bring about an additional decrease in oxygen affinity at physiological pH. Oxygen dissociation studies on the lysates showed a low oxygen affinity, a normal Bohr effect at 50% saturation, slightly decreased haem-haem interactions and a normal interaction with 2,3-DPG. Estimation of the percentage of Hb Hammersmith in the lysates of the patient's cells by selective precipitation of the abnormal beta chain with p-chloromercuribenzoate (PCMB) showed the presence of 30-33% abnormal haemoglobin. Assuming no interaction between the Hb A and the Hb Hammersmith, oxygen dissociation curves were calculated for Hb Hammersmith alone and these indicated that it has greatly decreased oxygen affinity, a normal Bohr effect, normal 2,3-DPG interaction and (somewhat) decreased haem-haem interactions.


Investigations on antithrombin III in normal plasma and serum.

Studies on antithrombin III (AT-III) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electrophoresis, AT-III from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT-III displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin III1 : IAT-III1) dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT-III2 and IAT-III3) became more evident. Normal human plasma and serum were filtered on Sephadex G-200 and the AT-III concentration measured in the fractions by rocket immunoelectrophoresis. The peaks of AT-111 were found in the same fractions for both plasma and serum and were coincident with the albumin peak of the plasma proteins. However, in the case of serum the AT-III concentration decreased less sharply in those fractions with higher molecular weight than in the corresponding plasma fractions. Analysis of these fractions by crossed immunoelectrophoresis revealed that the two components with slower electrophoretic mobility (IAT-III2 and IAT-III3) had higher molecular size than IAT-III1, that the concentration of IAT-III2 and IAT-III3 was significantly higher in serum, and that the high molecular weight components in plasma and serum were qualitatively identical. It is concluded that high molecular weight complexes between AT-III and activated coagulation factors may be present in normally circulating blood and that their detection and possibly quantitation can be achieved using the heparin/agarose crossed immunoelectrophoresis system.


Surface features of human eosinophils: a scanning and transmission electron microscopic study of a case of eosinophilia.

Cells were obtained from the peripheral blood of a patient with marked non-leukaemic eosinophilia. Transmission electron microscopy (TEM) showed typical mature eosinophils. The surface architecture of eosinophils by scanning electron microscopy (SEM) shows that most were spherical with varying numbers of microvilli; a smaller proportion of cells had ridge-like profiles, small ruffles and occasionally blebs. The surface features of the eosinophil thus resemble most lymphocytes and this finding must be considered when leucocyte populations are examined by SEM.


The endotoxin-induced coagulant activity of human monocytes.

Leucocyte suspensions, exposed to endotoxin in vitro, develop coagulant activity which has been identified as tissue factor. Pure suspensions of polymorphonuclear (PMN) neutrophils, lymphocytes and monocytes were exposed to endotoxin and tested for tissue factor activity after 4 h incubation at 37 degrees C. The results indicate that the monocyte is the cell primarily responsible for the endotoxin-induced coagulant activity of mixed leucocyte suspensions, the small amount of activity demonstrated in PMN neutrophil and in lymphocyte suspensions at high cell concentrations being accounted for by monocyte contamination of less than 1%.


The concentration dependence of the oxygen affinity of haemoglobin S.

The effect of the concentration of haemoglobin S (Hb S) on its oxygen-dissociation properties was studied using either reconstituted Hb-S cells of different mean corpuscular haemoglobin concentrations (MCHCs) prepared by osmotic lysis, or cells in which Hb S is diluted by the presence of another haemoglobin. Only 4% (phosphate buffer) and 21%(bis Tris) of the low oxygen affinity of fresh Hb-S cells was found to be due to their slightly elevated intracellular 2,3-DPG concentrations since when the cells were depleted of 2,3-DPG most of the low affinity remained. The low affinity showed a marked dependence upon haemoglobin concentration which was absent for 2,3-DPG-depleted Hb-A cells and, by extrapolation, the MCHC at which the oxygen affinities of the Hb-S cells became identical to that of the Hb-A cells was 14.5 g/dl in phosphate buffer and 13.1 g/dl in bis Tris. Both fresh and 2,3-DPG-depleted cells containing another haemoglobin as well as Hb S (Hb-SA, Hb-SC and Hb-SF cells) were also found to have low oxygen affinities provided that the intracellular Hb-S concentration(MC(Hb-S)C) was above a certain level. These also showed a strong dependence upon the MC(Hb-S)C. The mean MC(Hb-S)C at which the low oxygen affinities of the DPG-depleted cells were abolished were 8.3 g/dl (phosphate) and 11.2 g/dl (bis Tris). Hb F in fresh Hb-SF cells brought about a much greater increase in oxygen affinity than the same amount of either Hb A or Hb C. In 2,3-DPG depleted cells Hb A showed a greater ability to 'dilute' the Hb S than did Hb C. The conditions for the low oxygen affinity of Hb S were therefore found to be very similar to those required for the gelling of both pure Hb S, and Hb S in haemoglobin mixtures. It was concluded therefore that the low oxygen affinity of the Hb S was caused by the polymerization and that the difference between the oxygen affinities of Hb-S and Hb-A cells may be used as a measure of the polymerization process.


Haemoglobin C/alpha thalassaemia: haematological and biosynthetic studies.

A family with genes for haemoglobin C (Hb C) and alpha thalassaemia was studied. The mother had Hb-C trait. The father also had Hb-C trait but in addition displayed microcytosis, elevated Hb-F levels and a concentration of Hb-C less than usual for heterozygotes. The proband was homozygous for Hb-C but had Hb-F levels far exceeding those present in Hb-C disease. Biosynthetic studies of globin synthesis in both father and daughter showed a deficit of alpha chains relative to non-alpha chains, confirming the presence of alpha thalassaemia. The coexistence of alpha thalassaemia influences the level of mutant haemoglobin in haemoglobinopathies in which Hb C is present, in a fashion similar to that observed in sickle-cell trait.


G6PD hillbrow: a new variant of glucose-6-phosphate dehydrogenase associated with drug-induced haemolytic anaemia.

A new genetic variant of the red cell enzyme glucose-6-phosphate dehydrogenase is described. It was observed in a patient presenting with severe haemolytic anaemia and renal failure following ingestion of an overdose of Beserol (paracetamol and chlormezanone). The enzyme in the red cell had 12% of the activity of a normal B+ control, but only slightly lower activity in the kidney compared with a normal control. The red cell enzyme showed normal electrophoretic mobility and thermostability, a biphasic pH optimum curve, higher than normal utilization of the substrate analogues 2-deoxy-glucose-6-phosphate and deamino-NADP, and lower than normal Michaelis constants for both substrates, glucose-6-phosphate and NADP. The enzyme was strongly inhibited in vitro by high concentrations of paracetamol and chlormezanone. The extent of inhibition was similar to that for the enzyme from a normal B+ individual.


Ethanol inhibition of reticulocyte protein synthesis: the role of haem.

Ethanol, in concentrations of 0.05-0.8 M, inhibited intact human and rabbit reticulocyte protein synthesis in the presence of iron-transferrin for endogenous haem synthesis. Associated with this effect there was a conversion of polyribosomes to monoribosomes and a decreased incorporation of radioactive leucine into nascent globin chains. When physiological levels of ethanol (0.05-0.1 M) were used, these effects were prevented by incubation with 50 muM haemin and reversed by removing the alcohol and reincubating with iron-transferrin or haemin. The polyribosomal disaggregation was also prevented by stopping ribosomal movement with 5 mM cycloheximide. Neither ATP nor GSH levels were altered in the presence of ethanol. When non-physiological levels of 0.8 M ethanol were used, haemin did not prevent the inhibition of protein synthesis. Likewise, in the rabbit reticulocyte cell-free lysate system containing haemin inhibition was noted at concentrations greater than 0.05 M ethanol. The polyribosomal disaggregation in reticulocytes incubated with 0.8 M ethanol was associated with decreased dissociation of monoribosomes into subunits. Similarly, when ribosomes were directly suspended cell-free in 0.1 or 0.8 M ethanol there was a decreased percentage of subunits. These results indicate that physiological concentrations of ethanol inhibit initiation of reticulocyte protein synthesis secondary to a block in haem synthesis. When intact cells are exposed to high non-physiological concentrations of ethanol the inhibition is secondary to decreased ribosomal dissociation. The cell-free lysate inhibition is also through this effect on ribosomal dissociation. This study supports the view that alcohol is a direct toxin to developing red cell precursors via its effect on mitochondrial haem synthesis. The physiological role of the decreased dissociation of monoribosomes into subunits is not yet clear.


Determination of the packed cell volume using 131I-human serum albumin.

When trace quantities of labelled albumin have been added to blood, the packed cell volume (PCV) may be determined by isotope counting using the relationship: (plasma counts-blood counts)/(plasma counts). This method for measuring the PCV is not affected by plasma trapping between the red cells and the results do not depend upon the centrifugation conditions. The PCV determination with labelled albumin has a high precision (coefficient of variation = 0.9%) and there is very good agreement with the centrifugation PCV using Wintrobe tubes after this measurement has been corrected for plasma trapped between the red cells. No significant compression of the red cells occurred when the blood samples were centrifugated in Wintrobe PCV tubes.


The abnormal surface characteristics of the red blood cell membrane in congenital dyserythropoietic anaemia type II (HEMPAS).

The red blood cell membranes of patients with congenital dyserythropoietic anaemia Type II (CDA-II) were found to be abnormal. They had altered antigenic characteristics, decreased electrophoretic mobility, decreased sialic acid content, and abnormal filtration through polycarbonate filters. Proteins extracted from the CDA-II erythrocytes showed a different banding pattern on polyacrylamide gels compared to normal erythrocytes. Erythrocytes from clinically unaffected siblings and parents showed similar but less striking abnormalities of antigenic surface characteristics and banding patterns on polyacrylamide gels with nearly normal surface charge, sialic acid content, and filtration properties. These studies suggest a correlation between the degree of membrane abnormality and the clinical state of the CDA-II family member, demonstrate the erythrocyte in the heterozygote state is not normal, and support the concept of CDA-II as an autosomal recessive disease.


Observations on the ultrastructure of erythropoietic cells and reticulum cells in the bone marrow of patients with homozygous beta-thalassaemia.

An electron microscopic study of marrow fragments from patients with homozygous beta-thalassaemia has shown that 3% of early polychromatic erythroblast profiles and 20% of late polychromatic erythroblast profiles contain intracytoplasmic alpha-chain precipitates. Various nuclear abnormalities were found including the loss of parts of the nuclear membrane and the presence of intranuclear alpha-chain precipitates, and these abnormalities were virtually confined to the non-dividing, late polychromatic erythroblasts. As most profiles of the proliferating early polychromatic erythroblasts did not contain intracytoplasmic or intranuclear alpha-chain precipitates, it is suggested that the arrest of many of these cells in the G1 phase of the cell cycle may be related to the presence of an excess of free alpha-chains rather than to the presence of alpha-chain precipitates within them. The cytoplasm of the bone marrow reticulum cells contained early and late polychromatic erythroblasts at various stages of degradation, providing direct evidence of ineffective erythropoiesis.


The cellular basis for the defect in haemopoiesis in flexed-tailed mice. III. Restriction of the defect to erythropoietic progenitors capable of transient colony formation in vivo.

Three assays for erythropoietic progenitor cells have been applied to mice of genotype f/f and to nearly congenic +/+ controls. When f/f mice were tested for their ability to generate transient endogenous erythroid spleen colonies 4-6 days after 800 rads and 10 units of erythropoietin, the numbers of such colonies detected were greatly reduced, although normal numbers of spleen colonies appeared at later times (9-12 days) postirradiation. In contrast, cells capable of erythropoietic colony formation in culture (CFU-E) were present within the normal range in both f/f spleen and marrow and their sensitivity to erythropoietin in culture was the same as that found previously for CFU-E in the marrow and spleen of +/+ mice. Transfusion-induced plethora reduced the number of CFU-E in marrow to a similar extent in both f/f and +/+ mice; likewise, subsequent administration of 10 units of erythropoietin induced a rapid return in the number of marrow CFU-E in both genotypes. In the spleen, CFU-E numbers were approximately three-fold lower in f/f mice in each group. These results support the view that the 5 day assay for transient endogenous spleen colonies detects cells (TE-CFU) that are different from both CFU-E and pluripotent stem cells (CFU-S), although possibly overlapping to some extent with the immediate progenitors of CFU-E. The results also indicate that the generation or maturation of TE-CFU represents a primary site of expression of the f/f defect.


Pure red cell aplasia: studies on an IgG serum inhibitor neutralizing erythropoietin.

A new type of IgG serum inhibitor in adult pure red cell aplasia (PRCA) has been investigated. This inhibitor is directed against circulating erythropoietin (Ep) (PRCA type B), rather than the erythroid marrow (PRCA type A). Thus, the IgG inhibitor, after interaction with Ep in solution, is precipitated together with Ep by addition of goat anti-human gamma-globulins. Pre-therapy PRCA serum, although apparently devoid of Ep, shows considerable Ep activity following acidification and boiling. The inhibitor is absent from post-therapy serum, while Ep levels are elevated. An experimental model for PRCA type B has been established in normal mice after prolonged administration of pre-remission serum IgG.


Utilization of iron dextran in recurrent iron deficiency anaemia.

Previously published studies have documented a reduction in the rate at which iron stores laid down by iron dextran therapy can be utilized for haemoglobin synthesis after the acute demands of haemorrhage and phlebotomy. In order to determine if a defect in the mobilization of these stores exists in the face of a chronic stimulus to red cell production, 93 patients who had previously received a total dose infusion of iron dextran were examined for a recurrence of iron deficiency anaemia, and in those in whom anaemia had recurred, iron stores were assessed by marrow aspiration. Twenty of the 93 patients were found to have recurrent iron deficiency anaemia, and marrow aspiration in all failed to demonstrate stainable iron stores. Although the rate at which iron dextran can be mobilized from storage sites is reduced, the present study demonstrates that ultimately these stores are fully utilizable.


Ferritin synthesis in lymphocytes, polymorphs and monocytes.

A rapid two-stage method has been devised for the separation of different leucocyte populations from human blood. Different cell types can be obtained in an undamaged state and with little contamination. Ferritin and total protein synthesis has been determined by measuring [14C]leucine incorporation in culture media which contain varying amounts of added ferric iron or desferrioxamine. Both ferritin and total protein synthesis is greater in monocytes than in lymphocytes or polymorphs when the basal medium is used. Only monocytes show a consistent increase in ferritin production due to iron stimulation. Ferritin synthesis by monocytes, polymorphs and lymphocytes is inhibited in the presence of desferrioxamine.


A five year experience of the use of factor IX type DE(I) concentrate for the treatment of Christmas disease of Oxford.

A survey is presented of the use of the Oxford type DE(I) II-IX-X concentrate in the treatment of Christmas disease in Oxford from January 1970 to September 1974. 72 different patients were treated with a total of 2436 bottles of this concentrate from 143 different batches (each bottle containing 800-1000 units of factor IX). Although most doses were given for the treatment of minor haemorrhages into joints and muscles, 717 bottles of concentrate were used to treat 11 patients who underwent 14 major surgical operations. No episode of intravascular clotting or pulmonary embolism was seen in any patient receiving the concentrate. A detailed study of the plasma levels of factor V, VIII, total progressive antithrombin, platelets and fibrinogen degradation products was carried out before and after transfusion of type DE(I) concentrate in 14 patients. No significant alteration in those factors was found after the transfusion.


Alternative pathways for the activation of factor XIII.

Factor XIII is present in plasma as a proenzyme, which when activated catalyses the formation of epsilon(gamma-glutamyl)lysyl bonds in fibrin. In this study the activation of purified plasma factor XIII was examined quantitatively with the fluorescent amine incorporation assay. Activation products were examined by polyacrylamide gel electrophoresis. The serin proteases, thrombin, trypsin, chymotrypsin, and factor Xa, and also Reptilase were tested for their ability to activate factor XIII. Highly purified thrombins activated purified factor XIII; this reaction was not calcium dependent. Trypsin was also a potent activator, but no transglutaminase activity was found with chymotrypsin. The most highly purified preparations of Reptilase had no effect on factor XIII activity. Less purified Reptilase preparations activated factor XIII, which suggests the presence of another enzyme in these Reptilase preparations. Highly purified factor Xa was found to be an effective activator of purified factor XIII. In contrast to thrombin activation, this reaction required calcium. It may be that under certain circumstances factor XIIIa could be formed in vivo directly by the alternative pathway of factor Xa. Factor XIIIa could then crosslink fibrinogen, which would also provide an alternative pathway for thrombus formation. Also, the activation of factor XIII by both factor Xa and thrombin provides a further point of control in the blood coagulation process.


Polymerized monoclonal IgA in tuo patients with myelomatosis and hyperviscosity syndrome.

Two cases of multiple myeloma with IgA gammopathy and the syndrome of serum hyperviscosity are reported. A common feature of both patients was the production of large amounts of polymeric IgA (more than 50% of the monoclonal proteins existed as dimers or larger forms). However, similar large amounts of polymeric IgA were present in sera with only slightly raised viscosity, suggesting that factors such as the molecular configuration of the polymers or their sub-units may play a role in determining serum viscosity. The association of plasmapheresis and chemotherapy produced remarkable improvements of the symptoms attributable to increased serum viscosity, and 16 months after diagnosis one of the patients has survived relatively free of symptoms. The hyperviscosity syndrome is being diagnosed with increasing frequency in association with IgA multiple myeloma, and should be investigated in all cases with monoclonal proteins exceeding a concentration of 3-0 g/dl.


The blood group I and i antigens of amniotic fluid. I. Association of I and i antigens with blood group A, B and H antigens.

Human amniotic fluid has been shown to contain blood group i as well as I antigens. Crude extracts of amniotic fluids at 16-23 weeks of gestation were in general more active than those obtained at term. A pool of amniotic fluids which had A, B, H as well as I and i activity was fractionated with an insolubilized anti-I (Group 3 type) immunoadsorbent column. There was evidence for the occurrence of I and i determinants on macromolecules carrying A, B and H determinants, for the fraction specifically retained by the column was enriched by 50-60-fold in I and i activity and by at least 10-fold in A, B and H activity. In the fraction not retained by the column there remained the bulk of the A, B, H activity in addition to I activity of Group I type which is known to be distinct from the Group 3 determinant. With the aid of specific immunochemical fractionation procedures, human amniotic fluid should prove useful in structural studies of the several I and i determinants and of their relationship to other blood group determinants.


Auto-anti-Pra: a 'second' example in a newborn.

A second example of anti-Pra was recognized in a newborn infant which exhibited several signs of development disease. The antibody was complement-fixing 19S IgM and showed a high thermal range but no detectable haemolytic anaemia was associated. It gave negative reactions with a panel of animal cells as well as with two samples of human En(a-) cells. The latter, however, could absorb the antibody.


Ultrastructural studies of the bone marrow in sickle cell anaemia. I. The structure of sickled erythrocytes and reticulocytes and their phagocytic destruction.

Marrow aspirates from sickle cell patients were examined without prior deoxygenation and revealed the presence of a variable proportion (10-30%) of sickled red cells and reticulocytes. The main feature of sickled red cells was the presence of 17.6 nm fibres arranged in hexagonal order, and to a lesser extent in square or rectangular array, to form bundles of varying size and compactness which occupied the entire cytoplasm. The sickling pattern in reticulocytes was more variable. Some reticulocytes contained highly-ordered bundles of 17.6 nm fibres whose structure and distribution was identical to that in red cells from whiich they could be distinguished only by their content of organelles. Many reticulocytes exhibited less-organized fibre patterns ranging from localized aggregates to poorly-ordered regions of short fibres and filaments lacking apparent preferential orientation. In these cells, the bulk of the cytoplasm was not polymerized. Haemoglobin polymerization in reticulocytes led to entrapment of ribosomes in concentrated foci among the fibres. Frequently, iron-laden mitochondria were associated with sickled reticulocytes. The variation in pattern of sickling seen in reticulocytes is attributed to possible differences in concentration of Hb S. Correlative studies have shown that fibres were not induced by fixation procedures. Marked phagocytic degradation of sickled cells by macrophages was observed. The results are interpreted to indicate the possible intramedullary phagocytosis of red cells and reticulocytes, predisposed to sickling in the marrow. However, the data are also consistent with the removal of sickled elements from the circulating blood.


Abnormal membrane surface properties during maturation of rat reticulocytes elicited by bleeding as measured by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases is an extremely sensitive method for the separation of cells and for tracing subtle changes in the cells' membrane surface properties (primarily surface charge) as a function of in vivo processes (e.g. differentiation, maturation, ageing). Combining isotope labelling and cell countercurrent distribution techniques we have examined the membrane surface properties of rat reticulocytes produced in response to erythroid stress by bleeding. It was found that the rapid increase in the partition coefficient of normal reticulocytes subsequent to release into the peripheral blood (which reflects a rapid increase in membrane surface charge) is absent in reticulocytes produced in response to bleeding. In this way the reticulocytes behave like those produced in response to repeated phenylhydrazine injections. 'Stress reticulocytes' never mature to erythrocytes having normal membrane surface properties as judged by partition. The experiments show that our previous results are not due to the phenylhydrazine per se but rather to the production of 'stress reticulocytes'. Further, if remodelling of the phlebotomy-induced reticulocyte cell membrane occurs in the circulation as has been suggested it does not lead to a cell with normal membrane surface properties. Whether the abnormal membrane surface properties of 'stress reticulocytes' and of the erythrocytes to which they give rise affect the cells' life-span is not clear since phenylhydrazine-induced 'stress reticulocytes' have been reported to have at least a component of short-lived cells while the phlebotomy-induced cells are said to have a virtually normal life-span. It is likely that reticulocytes produced in severe anaemias, in general, mature to erythrocytes having abnormal surface properties (i.e. lower membrane surface charge).


Glucose-6-phosphate dehydrogenase variants from Italian subjects associated with severe neonatal jaundice.

Screening for the G6PD deficiency was carried out at the Maternity Division of the Galliera Hospital in Genoa, Italy. Two groups of subjects with hyperbilirubinaemia of non-immunological origin were examined: (a) 302 newborn babies of Sardinian extraction (on cord blood) and (b) 201 newborn babies of south Italian ancestry (on peripheral blood). Among 503 subjects, 43 showed an enzyme deficiency; in 39 the defect was of the Mediterranean type. In one case, previously described, the enzyme was of the A- type. In the remaining cases three different variants were identified. In the present work these three cases, each with severe neonatal jaundice, are reported. Their parents originated from Calabria, from Sardinia and from Sicily. The abnormal enzymes are respectively designated as GdDcbrousse-like,, GdGallura and GdAgrigento.


The significance of abnormal circulating cells in patients with Hodgkin's disease.

Buffy coat preparations of peripheral blood from two patients with Stage IVB Hodgkin's disease, 33 patients with Hodgkin's disease undergoing complete staging including laparotomy, 12 normal controls and eight patients with viral upper respiratory infections were examined for the presence of abnormal cells. Primitive histiocytic cells were seen in the two patients with IVB disease and in two patients with IIIB disease all of whom proved to be refractory to chemotherapy. Reed-Sternberg cells were seen in one IVB patient. Large cells with basophilic, agranular cytoplasm and moderately convoluted nuclei often with a perinuclear halo and nucleoli were detected in 30 of 35 patients with Hodgkin's disease, five of eight patients with viral infections but in none of 12 normal controls. Other abnormal large cells with round nuclei, occasional prominent nucleoli, grey to lightly eosinophilic cytoplasm and diffuse granulation were seen almost exclusively in Hodgkin's disease. There was no correlation between histology, stage, splenic involvement, skin test positivity and response to therapy with the presence or frequency of either of these cell types. This observation suggests that these cells are more likely to be 'reactive' rather than malignant. It is unusual for abnormal histiocytic cells to circulate in Hodgkin's disease and their presence may be a poor prognostic sign. The presence of other types of large atypical cells is not indicative of either haematogenous spread of Hodgkin's disease or of poor prognosis after treatment with radiotherapy alone.


The effect of diet and alcohol on the development of folate deficiency in the rat.

Studies of the rate of depletion of serum and tissue methylated and non-methylated folates were carried our in rats maintained for long periods on either a folate deficient (sucrose-water/sulphathiazole) diet or a deficient diet plus high alcohol intake. By means of implantation of a feeding gastrostomy tube, it was possible to sustain constant blood ethanol levels of between 50 and 300 mg/dl for 3-4 weeks with relatively normal calorie intake and without death of the animal. Using this animal model, which closely resembles severe alcoholism in man, a very rapid depression in serum 5-methyl tetrahydrofolate was observed similar to that reported in alcoholic man. At the same time, release of folates from liver stores was umimpaired by alcohol ingestion. Liver folate store depletion rates were identical for alcoholic and folate starved animals. The explanation for the sudden alcohol suppression of serum folate levels must, therefore, be sought at a point in the internal metabolic sequences of folate other than the delivery of folate stores to plasma.


Stimulation by propylthiouracil of the hexose monophosphate shunt in human polymorphonuclear leucocytes during phagocytosis.

The effect of propylthiouracil on glucose metabolism in human polymorphonuclear leucocytes was studied. At a therapeutically achievable concentration (0.1 mM), propylthiouracil stimulated hexose monophosphate shunt activity in normal leucocytes during phagocytosis but not in resting cells. However, in the presence of hydrogen peroxide it stimulated hexose monophosphate shunt activity in resting cells, and in the soluble fraction when reduced glutathione and reduced nictotinamide adenine dinucleotide phosphate (NADPH) were also present. Propylthiouracil had nor effect on glucose-1-C oxidation in either phagocytosing or resting leucocytes obtained from two male patients with chronic granulomatous disease. Stimulation of the hexose monophosphate shunt activity in normal leucocytes during phagocytosis also was demonstrated with methimazole, thiouracil and thiourea, but not with adenine, uracil or urea. There was an apparent minimal common structure requirement in thriourea. Propylthiouracil had no effect on phagocytosis, formate oxidation, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or catalase activities. Thus, the stimulation of the hexose monophosphate shunt activity by propylthiouracil is dependent on hydrogen peroxide and is best explained by its stimulation or participation in the glutathione cycle.


Brain iron: persistent deficiency following short-term iron deprivation in the young rat.

This study was designed to determine the content of non-haem iron in the brain as iron deficiency develops in the rapidly growing rat. Rats were provided with either an iron-deficient diet or an identical control diet with added ferrous sulphate starting at 10 d of age and continuing after weaning at 21 d. At 28 d or 48 d of age the deficient animals received 5 mg of iron (iron dextran) i.m. and were placed on the control diet regimen. The deficient animals had a concentration of non-haem iron in the brain that was 27% below the control value at 28 d and 22% below at 48 d. After 14-45 d of iron treatment, the non-haem iron remained depressed, 19-29% below the control means (P less tha 0.05 to 0.001). Ferritin iron in brain also remained depressed, 33-42% below the control means (P less than 0.01). In contrast, haematocrit, liver non-haem iron, and liver ferritin iron, although they were more profoundly depressed in the iron-deficient animals, promptly returned to control values after treatment with iron. Thus, a brief period of severe iron deficiency in the young rat resulted in a deficit of brain iron that persisted in the adult animal despite an adequate intake of iron.


Functional and metabolic studies of platelets from patients with Lesch-Nyhan syndrome.

Platelet function was investigated in three patients with the Lesch-Nyhan syndrome. Platelet count, morphology and size distribution was normal in all patients. Platelet turnover was normal. Electron microscopy did not reveal any ultrastructural abnormality. Template bleeding times were normal and prolonged after aspirin ingestion in two out of the three patients: the patient that failed to respond to the aspirin challenge also had decreased retention of platelets on a glass bead column. Biochemical studies revealed that total platelet ATP was reduced by 34% in the presence of a normal level of ADP in the storage pool. These platelets failed to incorporate radioactive hypoxanthine but did incorporate radioactive adenine to produce adenine nucleotides and a trace amount of guanine nucleotides. The results indicate that normal platelets have a functionally intact pathway for utilizing hypoxanthine as a source of preformed purine, and that the failure to salvage this purine, as in the Lesch-Nyhan syndrome, results in a decreased level of total platelet ATP. These findings suggest that platelets can function normally despite a one third reduction in total ATP content.


Heterogeneity of rabbit platelets. V. Preferential splenic sequestration of megathrombocytes.

Rabbits were either splenectomized or subjected to splenic blockade with phenylhydrazine in order to determine the splenic platelet and megathrombocyte (large-platelet) pools. The average splenic platelet pool calculated from both methods was 35% of total platelets. The average splenic megathrombocyte pool was 54% of total megathrombocytes. Adrenaline injection into rabbits or dogs revealed a rapid increase in both platelet count and megathrombocyte number which peaked at 2-6 min and returned toward normal in 5-10 min. The platelet volume distribution curve was shifted to the right, indicating the release of large platelets (megathrombocytes) into the circulation. The peak rise in megathrombocyte number was significantly greater than the peak rise in platelet count. Rabbits subjected to splenic massage shifted their platelet volume distribution curve to the right in the absence of a rise in platelet count. It is concluded that the spleen preferentially sequesters megathrombocytes.


The intercellular content of the three transcobalamins at various stages of normal and leukaemic myleoid cell development.

A study of the three serum transcobalamins (TC) in myeloproliferative disorders and acute leukaemia has suggested a granulocyte origin for the three binders; the later stages of this cell line might be releasing 'TC I' and 'TC III' while 'TC II' which is increased in sera from patients with acute myelogenous leukaemia, probably originates at least in part from myeloblasts. The intracellular content of each TC has been studied in normal and leukaemic bone marrow cells after separation on a discontinuous bovine serum albumin gradient. Myeloblasts contain a TC II-like protein almost exclusively but promyelocytes contain a TC I-like protein which increases as the cells mature whilst 'TC II' decreases. A TC III-like binder parallels 'TC I' but appears later and reaches a maximum in the polymorphonuclear cells. In various types of leukaemia, abnormal TC distributions are related either to marrow replacement by the blast cells or to a disorder of the maturation process in the leukaemic cell line.


Binding of vitamin B12--rat transcobalamin II and free vitamin B12 to plasma membranes isolated from rat liver.

When dialysed rat serum which contains a single, low molecular weight binder for vitamin B12, rat transcobalamin II (rat TC-II), was labelled in vitro with 57Co-vitamin B12 and then incubated at 30 degrees C (pH 7-5) with vesicles of highly purified plasma membranes separated from microsomal fractions of rat liver by density gradient centrifugation, the 57Co-vitamin B12-rat TC-II complex bound to high affinity sites on the vesicles via a specific (binding after correction for 'non-specific' binding in the presence of a large excess of the non-radioactive complex), saturable, and reversible interaction. The apparent affinity constant for the binding reaction was 5-5 X 10(9) M-1. Using the same incubation conditions, free vitamin B12 also bound to the vesicles of plasma membranes via a specific, saturable, but apparently irreversible interaction. Preincubation of the membranes with free vitamin B12 did not interfere with the subsequent binding of the vitamin B12-rat TC-II complex to the membranes; however, preincubation with the vitamin B12-rat TC-II complex did interfere, to some extent, with the subsequent binding of free vitamin B12. Dialysed rat serum, perhaps the free rat TC-II in the dialysed serum, also inhibited the binding of the vitamin B12-rat TC-II complex to the plasma membranes. The relationship of the binding sites identified in this report to the absorption of vitamin B12 by rat liver, and thus their physiological significance remains unknown until further work is done, perhaps using intact hepatocytes.


The anti-folate effect of methionine on bone marrow of normal and vitamin B12 deficient rats.

The effects of dietary vitamin B12 and methionine deficiency, and the in vitro addition of methionine, homocysteine, or folic acid on the methylation of dUMP to dTMP were studied in rat bone marrow culture. Vitamin B12 or methionine deficiency had no effect on the methylation reaction or on bone marrow folate levels although the vitamin B12 content in bone marrow was reduced in vitamin B12 deficiency. In vitro addition of vitamin B12 or folic acid also had no effect on the methylation of dUMP. In vitro addition of methionine reduced the methylation of dUMP and increased the proportion of 5-methyltetrahydrofolate at the expense of other folate coenzymes. The reason for this 'anti-folate' effect of methionine, which is the opposite to that found in liver, was not clear. The presence of 5,10-methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyltransferase were confirmed in rat bone marrow and they were inhibited by S-adenosylmethionine and methionine, respectively, in a similar fashion to that found with the liver enzymes. Homocysteine had no effect on the proportions of the various folate coenzymes in bone marrow but did inhibit the incorporation of deoxyuridine and deoxythymidine into DNA. It appeared that homocysteine exerted at a non-folate dependent step beyond the formation of dTMP.


Stimulation of erythrocyte glutathione reductase activity by flavin adenine dinucleotide (FAD) in Malaysian adults and newborns and their parents.

A study of glutathione reductase (GR) activity and its stimulation by flavin adenine dinucleotide (FAD) in erythrocytes of Malaysian newborns and adults of different racial groups showed that GR stimulation by FAD was greater than 20% in 50% of 866 newborns (57% of Malays, 55% of Indians and 41% of Chinese) and 54% of 274 adults (46% of Malays, 65% of Indians and 45% of Chinese). There was a significant negative correlation between GR activity and percentage FAD stimulation in both newborns and adults in all racial groups. Low GR activity and a high percentage FAD stimulation were more prevalent among parents of newborns with low GR activity than among parents of newborns with higher GR activity. Administration of riboflavin to mothers with low GR activity resulted in increased GR activity and a decreased percentage of FAD stimulation. None of the individuals examined had clear clinical manifestations of riboflavin deficiency. It is concluded that subclinical riboflavin deficiency leading to low GR activity is prevalent in Malaysia among adults and newborns, especially among Malays and Indians.


Effects of ADP and ATP on bovine fibrinogen- and ristocetin-induced platelet aggregation in Glanzmann's thrombasthenia.

With thrombasthenic platelets, aggregation induced by ristocetin (R) and bovine fibrinogen (BF) is reversible, provided ATP is not added to the system. ADP inhibits BF and R induced aggregation, and when added after the onset of aggregation causes rapid disaggregation. ATP prevents these inhibitory and disaggregation effects of ADP. A mechanism is suggested whereby co-operative effects occur between receptors in the membranes of thrombasthenic platelets.


Leucocyte alkaline phosphatase as a marker of cell maturity: a quantitative cytochemical and autoradiographic study.

A sensitive and reproducible cytochemical technique for the demonstration of leucocyte alkaline phosphatase (LAP) has been evaluated. It produces a stable reaction product which can be readily quantified microphotometrically. In the circulating neutrophil polymorphonuclear leucocytes of rats the mean LAP levels measured in this way fluctuated only slightly from day to day. A highly significant correlation was demonstrated between LAP levels of neutrophils and their age, determined by isotope labelling with tritiated thymidine and by nuclear morphology. The correlation between the age of neutrophils and the complexity of their nuclear morphology has also been confirmed. The technique provides a useful tool for the study of leucocyte kinetics in disease states.