Detection of red blood cell antibodies using bacterial antiglobulins: a new assay [abstract]

OBJECTIVE: To develop an agglutination technique using Staphylococcal protein A (SpA) and Steptococcalprotein G (SpG) to detect human red blood cell antibodies. METHODS: Blood samples were obtained from the National Transfusion Service of Jamaica. SpA, SpG, and anti-IgG, -C3d were commercial preparations. SpA and ApG were incubated with sensitized human red blood cells (RBC) to assess their RBC agglutinating capacity. The Ouchterlony technique was used to determine binding between the bacterial antigens and the IgG in human serum. Polyethyleneglycol (PEG) was used as an agglutination enhancer. Agglutination techniques for a large number of samples were developed, using 96 percent well polystyrene microplates and a microscope for visualizing the agglutination techniques to detect human anti-RBC IgG was compared with the traditional Coombs' test. Sensitivity and specificity were determined. RESULTS: SpA and SpG did not appear to cause agglutination of the red cells sensitized in vivo and in vitro. However, no precipitation bands were formed between human serum and the supernatant obtained after reaction of the sensitized RBC with SpA and SpG (Ouchterlony technique). These results indicated that indeed there was binding of SpA and SpG with the sensitized cells since they were not available for binding with human serum. In additon, SpA and SpG agglutinated the sensitized red blood cells in the presence of the PEG. When compared to the Coombs' test, the following results were obtained with new techniques. For the direct method, sensitivity was 93.8 percent and 95.1 percent for SpA and SpG respectively (n= 81), and specificity was 91.4 percent and 93.5 percent for SpA and SpG, respectivley (n= 93). For the indirect method, sensitivity was 96.3 percent and 97.5 percent for SPA and SpG, respectively (n= 81) and its specificity was 100 percent for both proteins (n= 85). CONCLUSION: Agglutination techniques using SpA and SpG constitute alternative and feasible tests for the detection of human red blood cell antibodies. (AU)

The role of labelled red blood cell scintigraphy in the detention of acute gastrointestinal bleeding

This paper describes the experience with 99m Technetium labelled red blood cell (99mTc RBC) scintigraphy in twenty-two patients presenting with acute gastrointestinal (GI) tract bleeding. Studies were postitive in thirteen cases - eight from the lower GI tract and five from the upper. The data from surgical intervention were available in ten cases. Scintigraphy cannot diagnose the cause of GI bleeding, as it is a nonspecific study. Its usefulness lies in its ability to accurately diagnose the bleeding site, as was shown in this study where there was good surgical correlation. The simplicity, reproducibility and reliability of the technique, particularly when bleeding rates are low and intermittent, make it, in our view, the first line of investigation in any patient with suspected bleeding from the colon or upper GI tract if endoscopic evaluation is not possible in the latter. Its current under-utilisation in the Caribbean may be a reflection of the lack of nuclear imaging facilities.(Au)

Decreased insulin binding to erythrocytes and mononuclear leucocytes in normal dogs administered with S-nitroso- glutathione

Nitric oxide is a pathogenic factor of inflammatory islet cell death in type 1 diabetes. An early event in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) is intraislet accumulation of activated macrophages. Their secretory products such as nitric oxide (NO) are found to play a crucial role in islet destruction. Macrophage activity and progressive islet cell destruction persist during a long period of chronic inflammatory events preceding diabetes, suggesting the presence of nitric oxide contributing to continued immunostimulation. It has been shown previously that the nitric oxide donor, S-nitrosoglutathione (GSNO) caused persistent postprandial hyperglycaemia in normal healthy dogs at 35 and 50mg/kg. parallel with an increase in plasma nitrate concentration and decrease in insulin secretion. This study was designed to investigate differences in cellular binding of insulin in dogs administered with the GSNO. A time course assay of insulin binding, to isolate erythrocytes and mononuclear leucocytes from dog administered with GSNO, was done during the oral glucose tolerance test (OFTT). The erthrocyte receptor assay performed was the methodology used by Ghambir et al (1977). A modification was also done for mononuclear leucocytes insulin binding assay. The plasma glucose levels were measured by the glucose oxidase method, while the insulin levels were determined by radio-immunoassay. The results of these studies show that erythrocytes and mononuclear leucocytes from dogs administered with GSNO have decreased ability to bind insulin when compared to erythrocytes and mononuclear leucocytes from the controls. In dogs administered with GSNO, there was less binding of erythrocytes and mononuclear leucocytes, 44 percent and 29 percent respectively, when compared to the bound free ratio value of the controls. The data also shows that erythrocytes and mononuclear leucocytes from dogs administered with GSNO have 256 and 10.7x10 insulin receptor sites per cell, respectively, compared with 296 and 18.4x10 per cell for the control (P<0.05). Competitive inhibition studies using unlabelled insulin indicate that the affinity of insulin for its receptor on erythrocytes from dogs administered with GSNO was also significantly different from the controls, while that of mononuclear leucocytes from both group was comparable.(AU)

Postural vasoconstriction and leg ulceration in homozygous sickle cell disease

Chronic leg ulceration is a major cause of morbidity in patients with homozygous sickle cell disease; the ulcers commonly resolve on bed rest. We have therefore compared the cutaneous vascular response to dependency in three groups of eight patients with sickle cell disease (those with an active ulcer, with an ulcer scar and with no history of ulceration) and in eight subjects with normal haemoglobin and no history of leg ulceration. We monitored with a laser Doppler flowmeter, the change in red cell (erythocyte) flux induced in the skin of the leg, at two sites proximal to the malleoli, with the leg horizontal and 5 and 10 min after moving the leg to the dependent position. With the leg horizontal, mean cutaneous red cell flux was was substantially higher in normal cells of patients with sickle cell disease than in normal subjects and was higher still at the site of the ulcer scar. On dependency, red cell flux fell not only in normal subjects but also in the patients with the sickle cell disease. The fall in red cell flux in normal skin of patients with sickle cell disease was smaller than in normal subjects when considered as a percentage of the control values, but in absolute terms the falls in red cell were similar in sickle cell patients normal subjects. By contrast, the fall in red cell flux at the ulcer/scar site was greater than in normal skin from sickle cell patients. We propose that high resting perfusion is important in patients with sickle cell disease to maintain normal integrity of cutaneous tissue and that pronounced vasoconstriction on dependency hinders the healing and encourages recurrence of the leg ulcers (AU)

Glyoxalase I activity in erythrocytes from severely malnourished children

The enzyme glycoxalase I (glyox I) is involved in metabolic detoxification, and requires glutathione (GSH) as a cofactor. Given the low concentration of whole blood GSH in children with oedematous malnutrition, it is possible that the function of this pathway may be compromised in these children. Glyox I activity was therfore assayed in erythocytes taken from 133 severely malnourished children and 21 age-matched controls. The mean values (ñSEM) for the marasmic group (marasmus: 105 ñ 4/u/gm Hb) and the group with kwashiorkor (Kwash: 103 ñ 4/u/gm Hb) were not significantly different from controls (cont: 104 ñ 2u/gm HB)>. In the group with marasmic-kwashiorkor (M-K: 88 ñ 4u/g Hb) Glyox I activity was significantly lower in controls (p < 0.005), as well as in children with marasmus (p < 0.005), and kwashiorkor (p < 0.05). Enzyme activity was lower than normal in 45 percent of the MK group. Seven children died subsequent to admission; in five cases Glyox I activities were exceedingly low. There was a weak positive correlation between Glyox I activity and whole blood levels of GSH (r=0.215). We conclude that Glyox I activity is relatively unaffected in malnutrition, except in those with M-K and especially those who do not survive the acutely malnourished state (AU)

Daily variability in resting levels of cardiovascular variable in normal subjects and those with homozygous sickle cell disease

Measurements were made of cardiovascular variables and oral temperature in 16 male subjects with homozygous sickle cell disease (SS) and in 17 matched controls (AA) at 10.00 a.m, 1.00 p.m and 4.00 p.m. All subjects were in a rested state throughout. At 10.00 a.m. mean arterial pressure was lower, while heart rate, total forearm blood flow and cutaneous red cell flux in the forearm were higher in SS than AA. Vascular resistance in total forearm and forearm skin, calculated by dividing arterial pressure by blood flow or red cell flux, were lower in SS but hand cutaneous red cell flux and vascular resistance were not significantly different in SS and AA. In both SS and AA, there were parallel increase over the three sessions, in mean arterial pressure (by approximately 12 and 10 percent respectively) forearm vascular resistance (by approximately 17 and 27 percent) and hand cutaneous vascular resistance and hand cutaneous resistance (by approximately 2240 and 350 percent) whereas forearm blood flow and hand cutaneous red cell flux fell. By contrast, forearm cutaneous resistance showed no change during the day in SS, but increased progressively in AA (by approximately 75 percent). These results indicate that, during the day, there is progressive vasconstriction in forearm muscle and hand skin in SS and AA and also in forearm skin of AA that contributes to a progressive rise in the resting level of mean arterial pressure. We suggest this daily variability should be considered in studies of cardiovascular function: within a given study they should be performed at the same time of day.(AU)

á-N-terminal glycated haemoglobins (HbX) in subjects with common haemoglobinopathies: relation with fructosamine and mean erythrocyte age - abstract

á-N-terminal glycated haemoglobins (HbX, by HPLC), serum fructosamine and erythrocyte polyamines were determined in non-diabetic adults with HbAA, HbAC, HbAS, HbCC, HbSC, HbSS and Hb/HPFH. The groups did not differ in fructosamine. Mean (95 percent confidence limits) percentage HbX were: 4.4 (range 4.1 - 4.8) for HbA in HbAA, 4.3 (range 3.9 - 4.8) for HbA in HbA in HbAA, 4.3 (range 3.9 - 4.8) for HbA in HbAC, 4.1 (range 3.6 - 4.6) for HbC in HbAC, 4.4 (range 4.0 - 4.7) for HbA in HbAS, 2.6 (range: 2.3 - 3.8) for HbC in HbCC, 2.0 (range 1.5 - 2.4) for HbS in HbSC, 0.9 (range: 06. - 1.3) for HbS in HbSS, and 1.3 (range: 0.8 - 2.4) for HbS in HbS/HPFH. Considering all data, there was a non-linear inverse relation between HbX and erythrocyte polyamines, indicating that percentage HbX decreases with decreasing mean RBC-age. It is concluded that HbX in subjects with heterozygous haemoglobinopathies is to be expressed as percentage HbX + HbC, not total haemoglobin. Interpretation of HbX in subjects with decreased RBC half-life is difficult. Fructosamine seems a suitable alternative (AU)

Low sickle-cell linoleic acid content is related to young erythrocyte age - abstract

Erythrocyte (RBC) fatty acids (FA) and polyamines were determined in subjects with HvM (n=29), HbAC (3), HbAS (41), HbSC (25) and HbSS (19). FA of plasma cholesterol esters (CE), plasma phosphatidylcholines (PC), RBC PC, and plasma and RBC PC-species were studied in subgroups. RBC of patients with HbSS and HbSC had abnormal FA, PC-FA, PC-species and polyamines. There were no major differences in plasma CE-FA, PC-FA and PC-species. Low 18:2U6 in RBC, RBC PC and RBC PC-species of patients with HbSC and HbSS are related to RBC polyamines. Low RBC 18:2U6 is almost stoichiometrically compensated for by higher stearic and palmitic acids. Circulating RBC from patients with HbSC and HbSS have normal total polyunsaturated FA with 20 carbons or more. Low RBC 18:2U6 of patients with HbSS and HbSC is rather related to young RBC-age (RBC polyamines) than to diet (plasma CE-FA). Their rapid RBC turnover causes incomplete RBC-FA exchange with plasma species (AU)

Elevated glutathione S-transferase activity in erythrocytes from malnourished children

Glutathione S-transferases (GSTs) are principally involved in detoxication. These enzymes can be induced by an increased flux of substrate, such as occurs during pro-oxidative stress or antioxidant deficiency. We tested the hypothesis that the postulated oxidative stress in severe malnutrition would result in induction of GSTs in erythocytes. Erythrocyte GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was measured in 271 malnourished children (22 undernourished; 92 marasmic; 82 kwashiorkor; 75 marasmic-kwashiorkor) and 48 healthy children. GST activity in the malnourished children was significnatly higher than the control group (p < 0.01). The GST activity in the four classes of malnutrition did not differ. There was a weak relationship between GST activity and the height deficit, but not with the weight deficit, or the clinical features displayed by the children. The 11 children that died had a higher value than the survivors. There was no change in GST with anthropometric recovery. We conclude that erythrocyte GST has been induced in children with malnutrition. Induction of erythrocyte GST may be the result of exposure of the children to oxidative stress during the months prior to their presentation with severe malnutrition (AU)

Haemorrheological disturbance in diabetes mellitus - abstract

Diabetes mellitus is one of several metabolic diseases in which haemorrheological disturbances have been observed. In diabetes, particularly of long duration, there are marked alterations of the microcirculation. These changes are associated, at least in part, with rheological disturbances. Diabetic microangiopathy affects the retina, the enal glomeruli, the nerves and probably the entire network of capillaries. Rheological studies in both insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetics have shown a decrease in red cell deformability, an increase in red cell aggregation and plasma fibrinogen concentration. These alterations are aggravated by concurrent hypertension and the severity of the disease. The combined effect of these changes is the development of a hyperviscosity syndrome in the diabetic patient. The clinical significance of diabetic hyperviscosity is a marked impairment in blood-flow, in particular, across the microcirculation; hence diminished tissue perfusion (AU)

Indicators of long chain polyunsaturated fatty acid status of exclusively breastfed infants at delivery and after 20-22 days

The fatty acid composition of plasma cholesterol esters (CE), erythrocytes (RBC) and mature milk from seven lactating women and their exclusively breastfed newborns, living on Dominica, were studied. Blood samples were taken from umbilical cord and mother at birth. A sample of breastmilk was collected on day 20-22 postpartum, together with a blood sample from the baby. At birth, cord blood plasma CE and RBC total long chain polyunsaturated fatty acid (LC-PUFA) contents were higher, and linoleic (18:2c, omega 6) and alpha-linolenic (18:3c, omega 3) acid contents lower, than in corresponding maternal compartments. Cord blood RBC LC-PUFA omega 3 content was lower and LC-PUFA omega 6 content higher than in maternal RBC. After birth, feeding with human milk led to a drop in LC-PUFA content in the plasma CE fraction, whereas RBC LC-PUFA content remained virtually constant. Current understanding of the origin and relative affinity of fatty acids incorporated in plasma CE and RBC suggests that RBC LC-PUFA content is a more reliable parameter for LC-PUFA status than plasma CE LC-PUFA content. The RBC LC-PUFA data suggest therefore that at birth the newborn has a lower LC-PUFA omega 3 status than the mother, and that this does not change during three weeks of exclusive breastfeeding (AU)

Long chain polyunsaturated fatty acid status of the exclusively breastfed infant in Dominica at delivery and after 20-22 days - abstract

The fatty compositions of plasma cholesterol esters (CE) and erythrocytes (RBC) from five lactating women and their exclusively breastfed newborns, living in Dominica, were studied. Blood samples were taken from the umbilical cord and mother at birth. A second blood sample of the newborn and a sample of breast milk were taken on day 20-22 postpartum. Fatty acids were determined by capillary gas chromatography. At birth, cord blood plasma CE and RBC long chain polyunsaturated fatty acids (LC-PUFA) contents were higher, and linoleic acid (18:2c, w6) contents lower, than in corresponding maternal compartments. Accretion of LC-PUFA by the foetus may be accomplished by a-fetoprotein , that has a high affinity for LC-PUFA and is taken up by a variety of foetal tissues in a receptor-mediated fashion. After birth breastfeeding leads to a drop of LC-PUFA content in the plasma CE fraction, whereas RBC LC-PUFA content remains constant. Is is conceivable that RBC LC-PUFA content is a more reliable parameter for LC-PUFA status than the plasma CE LC-PUFA (AU)

Profile of plasma and erythrocyte enzymes: whole blood 2, 3 bisphosphoglyceric acid and reduced glutathione levels in Jamaican diabetics - abstract

The metabolism of normal red cell is well adapted to protect itself from oxidative stress. The maintenance of red cell membrane, haemoglobin and enzymes containing sulfhydryl groups (-SH groups) are dependent upon the levels of reduced glutathione (GSH). Alteration in the activities of enzymes such as glutathione reductase and glutathione peroxidase will affect the levels of GSH and consequently the red cell metabolism. Acetylcholinesterase, an SH-dependent enzyme, also increases in the red cells in diabetics and is related to membrane fluidity. The level of 2, 3- Bisphosphoglycerate (2,3-BPG) which serves an important mechanism by which the body regulates its oxygen supply from haemoglobin to meet tissue demands, also increases in chronic anaemias observed in diabetics. In the present studies the activities of plasma and erthrocyte cholinesterase, and erthrocyte glucose-6- phosphate dehydrogenase were measured in 40 diabetic patients (both IDDM and NIDDM groups). The whole blood 2, 3-BPG and GSH were also estimated in these patients. All five parameters showed significant increases in diabetic as compared to non-diabetic controls. The results could point to a potential role of G6PD 2, 3-BPG GSH and choline sterase as indicators of diabetes control (AU)

The effect of malnutrition on insulin binding to rat erythrocytes

Insulin binding to erythrocyte receptors was compared in malnourished and control rats. Percentage specific insulin binding to malnourished rat erythrocytes was significantly lower than to control erythrocytes (P<0.001). The low insulin binding in the malnourished rat erythrocytes was accompanied by low insulin receptor affinity (P=0.035).(AU)

Insulin receptor studies of erythrocytes and mononuclear leucocytes in phasic insulin diabetes mellitus

This study was designed to investigate any differences in cellular binding of insulin between phasic insulin-dependent (malnutrition-related) diabetes mellitus (PIDDM) and insulin-dependent, non-insulin-dependent, and normal controls. Isolated, washed red and white blood cells obtained after 12-14 hr fast, were separately incubated with varying concentrations of non-radioactive insulin, and a fixed quantity of radioactively labelled insulin. After the 3-hr incubation, cells were washed with buffer, and radioactivity determined on an autogamma counter. Percentage binding, receptor sites number an affinity were all determined by linear regression of the Scatchard plot. Fasting plasma insulin and glucose levels were also assayed. The results obtained showed decreased binding of insulin in red blood cells (11.3 ñ 1.3 percent) and white blood cells 2.9 ñ 0.5 percent) in PIDDM. This was due to decreased receptor sites (red blood cells 39 ñ 11; white blood cells 0.5 ñ 0.11 x 10 to the 4th) as well as decreased affinity (red blood cells 0.14 ñ 0.03 x 10 to the 9 M to the -1) when compared to the normal and diabetes (malnutrition-related diabetes mellitus) is characterized by decreased red and white cellular binding to insulin, in addition to decreased production of insulin.(AU)

Reduction in vitro of red cell glutathione reproduces defects of cellular sodium transport seen in oedematous malnutrition

Red cells in oedematous malnutrition (kwashiorkor) have an increased sodium content, 'leakiness' to sodium and enhanced sodium pumping. In non-oedematous malnutrition (marasmus) there is a reduction in the sodium pump activity. The explanation has hitherto been unknown but the glutathione content of red cells is low in kwashiorkor and normal in marasmus. We artifically lowered the glutathione content of normal red cells to values characteristic of mild oedematous malnutrition, using the enzyme inhibitors bischloronitrosourea (BCNU) and buthionine sulfoximine (BSOX). After preincubation, the cells were washed to remove the inhibitors and oxidized glutathione. Cellular content of sodium and potassium, and 86Rb influx were then measured. The reduction in glutathione reproduce the abnormalities of sodium content and flux observed in kwashiorkor. We suggest that oxidant stress in kwashiorkor, by reducing cellular glutathione, may affect cell membrane electrolyte transport. This may act through alterations in membrane sulfhydryl groups. Glutathione depletion may therefore play an important role in the clinical picture and natural history of oedematous malnutrition and may have relevance to other conditions where oxidant stress occurs. (AU)

Metabolism of phosphoinositides in sickle-cell anaemia erythrocytes - abstract

Polyphosphoinositides (PPI), minor components of red cell membrane phospholipids, undergo a rapid turnover of their phosphomonoester groups, but the role of the turnover has not been established definitely. It has been suggested that, in the red blood cell, phosphatidylinositol 4, 5-biphosphate (PIP2), and to a lesser extent, phosphatidylinositol 4-phosphate (PIP) are involved in the molecular interactions between skeletal and integral proteins and may play a part in cell shape and volume regulation. Since sickle-cell anaemia erythrocytes (SS cells) are known to have multiple structural and functional anomalies, particularly abnormal cell shape and volume regulation, we have investigated PPI metabolism in these cells. Concentrations of PPI and the 32P incorporation into these phospholipids have been measured in intact SS cells or isolated membranes, with AA cells as controls. The concentrations of phosphatidylinositol (PI) and PIP2 were the same in AA and SS cells. In contrast, major differences were found in the content of PIP which approximately doubled in SS cells. There was markedly higher incorporation of 32P in PPI of SS than AA cells after incubation with 32P (Pi). This increased turnover of PPI could be explained by a modification of the enzymes (kinases and phosphatases) involved in the metabolism of these phospholipids. The activities of the PI/PI 4-phosphate kinases have been studied in isolated membranes. The synthesis of PIP and PIP2 was increased in SS as compared to AA membranes. Further experiments are needed to elucidate the mechanism for this increased synthesis (AU)

Blood lead and zinc protoporphyrin levels in donkeys and mules near a secondary lead smelter in Jamaica

During the course of an investigation into community lead poisoning near a secondary lead smelter in Jamaica, blood lead and zinc protoporphyrin levels were measured in 8 exposed and 6 (3 Jamaican, 3 US) unexposed donkeys and mules. The blood lead levels of 6 animals in the contaminated area ranged from 7.5 to 33 ug/d1 (mean=17.6 ug/d1), compared to 1.8 and 2.4 in unexposed Jamaican animals. More striking was the difference in zinc protoporphyrin levels; all 8 exposed donkeys and mules had values between 900 and 1890 ug/d1, compared with a range of 34-46 ug/d1 for 3 Jamaican control donkeys. These findings suggest that zinc protoporphyrin may be a useful method of screening for subclinical lead toxicity in equines (AU)

Changes in red cell insulin receptors during recovery from severe malnutrition

Red cell insulin binding was studied in 13 Jamaican children (age range 4-24 months), while malnourished (MAL), during early recovery (GI), late recovery (GII), and after anthropometric recovery (REC). The rate of weight gain (RW), the energy intake (EN), and the protein intake (PR) were monitored at each phase of the study. Four-hour fasting blood samples were used, and the insulin binding characteristics were investigated in the physiological range of insulin concentrations (16.7-1670 pM). Analyses of variance were used to examine differences in the variables measured at the four phases. Red cell-specific insulin binding (SB) was lower in MAL than in GI (P<0.001) and in (GII) (P=0.026). SB in REC and MAL were not significantly different. Insulin receptor affinity (K) was also lower in MAL than in GI (P<0.001), GII (P<0.001), and REC (P=0.012). The insulin receptor number (S) appeared to be high in malnutrition and to decrease as recovery progressed; however, the decrease was not significant. Children with fever demonstrated high insulin binding. Plasma insulin (IN) rose during recovery, and was significantly higher in GII than in MAL (P=0.01). There was no difference in plasma glucose (G) at any phase of the study. The interrelationships among the variables measured were investigated longitudinally using multiple regression analyses. SB was positively associated with S (P=0.032), EN (P=0.029), and PR (P=0.0076). S was negatively associated with K (P<0.001). The associations of S and K with PR were positive and approached significance (P = 0.09 and P = 0.07 respectively). RW was positively associated with PR (P<0.001), and with EN (P=0.001). There were no significant relationships between G and any of the other variables longitudinally. However, correlations of the variables within phases demonstrated that in MAL, G was negatively associated with SB (P<0.05) and with K (P<0.05); but in REC, G was positively associated with SB (P<0.05). These results demonstrated that in severe malnutrition, the red cell insulin receptor affinity was low. During catch-up growth when protein and energy intakes were increased, both insulin receptor affinity and specific insulin binding were also increased. The negative relationship between insulin binding and plasma glucose during malnutrition may be related to carbohydrate intolerance (AU)