Comparison of three homogeneous methods for measuring high-density lipoprotein cholesterol with a precipitation method in diabetic and non-diabetic subjects.

Three homogenous methods for the automated measurement of high-density lipoprotein-cholesterol (HDL-C) are studied and compared with a precipitation method in diabetic and non-diabetic subjects. All three methods meet the precision criteria of the National Cholesterol Education Programme (NCEP). Triglycerides did not cause significant interference up to 10 mmol/L in the immuno-inhibition method (Wako) and up to 40 mmol/L in the Randox and Roche methods. Haemoglobin, up to a concentration of 5 g/L, had only negligible effect on the performance of all three homogeneous methods. Bilirubin caused an increasing positive bias in all methods above a concentration of 50 micromol/L. In comparison with the precipitation method, the new homogeneous methods agreed for type 1 diabetic patients but showed a positive bias for the control subjects and patients with type 2 diabetes. The bias of HDL-C levels in type 2 diabetes may be sufficient to affect the calculation of cardiovascular risk, and may therefore influence the decision to prescribe lipid-lowering medication.

Severe hyponatraemia: investigation and management in a district general hospital.

AIMS: To study the incidence, investigation, and management of severe hyponatraemia (serum sodium < 120 mmol/litre) over a period of six months in a district general hospital. METHODS: The laboratory computer was used to identify all inpatients who had a serum sodium concentration of less than 120 mmol/litre over a six month period. The records of these patients were reviewed for the relevant demographic, clinical, and laboratory data, in addition to diagnosis, treatment, and outcome of hospitalisation. RESULTS: Forty two patients were studied, with a female to male ratio of 2 : 1. Nine patients had central nervous system symptoms, and four of these patients died in hospital. Only 14 patients had their urinary electrolytes and/or osmolality checked. A diagnosis of syndrome of inappropriate secretion of antidiuretic hormone (SIADH) was mentioned in eight patients, sometimes without checking their urinary electrolytes or osmolality. Twenty one patients died in hospital. The patients who died did not have lower serum sodium values or a higher rate of correction of hyponatraemia, but they all suffered from advanced medical conditions. CONCLUSIONS: The possible cause of hyponatraemia should always be sought and that will require an accurate drug history, clinical examination, and assessment of fluid volume, plus the measurement of urinary electrolytes and osmolality in a spot urine sample. The diagnosis of SIADH should not be confirmed without the essential criteria being satisfied. The current or recent use of diuretics is a possible pitfall in the diagnosis of SIADH. The rate of serum sodium correction of less than 10 mmol/day is probably the safest option in most cases.

Comparison of two direct methods for HDL cholesterol measurement with an indirect precipitation method in diabetic patients.

The conventional precipitation method for measuring HDL cholesterol involves a centrifugation step which prevents automation of the method. Several methods have been introduced for measuring HDL cholesterol without the need for a centrifugation step. These new methods are therefore automatable and can process a large number of samples in a short period of time. Measuring HDL cholesterol is an important aspect of management of diabetes mellitus. In this study, we compared 2 direct methods for measuring HDL cholesterol with a conventional precipitation technique in 63 patients with either Type 1 or Type 2 diabetes mellitus. Both direct methods showed acceptable precision but they both showed positive bias compared to the conventional precipitation method. The greatest degree of bias occurs at low HDL cholesterol levels, which are more important for Type 2 patients. Such differences may affect cardiovascular risk calculation in patients with diabetes. Further studies are required to investigate if a correction factor needs to be introduced when these direct assays are used to measure HDL cholesterol in patients with Type 2 diabetes mellitus.

Serum fructosamine as a marker of 5-year risk of developing diabetes mellitus in patients exhibiting stress hyperglycaemia.

AIMS: We examined whether the level of random serum glucose (RSG) in subjects exhibiting stress hyperglycaemia is a useful marker of the future risk of developing diabetes mellitus (DM), and whether serum fructosamine is of any additional value. METHODS: All non-diabetic adults attending Accident and Emergency in 1994-1995, who had venesection, were studied. Serum fructosamine and RSG were routinely measured in all such patients. Using the laboratory biochemistry database the number of subjects with stress hyperglycaemia (RSG > 11.1 mmol/l) was determined, and their corresponding fructosamine values were recorded. The number of subjects who developed DM over the following 5 years was determined. RESULTS: Three hundred and seventeen patients had stress hyperglycaemia, and follow-up data were available on 224 patients. Of these patients, 63 (28%) had developed DM over the 5 years follow-up period. RSG and fructosamine levels at baseline of patients subsequently developing DM were (mean +/- sd (range)) 16.7 +/- 7.0 (11.2-55.0) mmol/l and 3.3 +/- 0.6 (1.3-4.5) mmol/l, respectively. The patients who did not develop DM had a similar baseline RSG, 15.9 +/- 3.3 (11.2-30.6) mmol/l; P = 0.170, but lower baseline fructosamine, 2.4 +/- 0.4 (1.6-3.8) mmol/l; P < 0.001. Receiver-operating characteristics showed that a serum fructosamine > or = 2.8 mmol/l was a useful marker of the future risk of DM (75% sensitivity, 74% specificity, 53% positive and 88% negative predictive power). CONCLUSIONS: The level of RSG in stress hyperglycaemia does not predict the future development of DM. Raised serum fructosamine is a more useful marker of future DM risk than RSG alone. Further prospective studies are needed.

Characterization of corticotropin-releasing hormone binding sites in the human placenta.

The human placenta synthesizes and secretes large amounts of corticotropin-releasing hormone (CRH) which has been implicated in the triggering of parturition. The placental CRH was found to act in a paracrine manner to stimulate secretion of ACTH and beta-endorphin. In view of this we sought to characterize CRH binding sites in the human placenta. The specific binding of 125I-tyrosyl-ovine CRH (125I-oCRH) to placental membranes was dependent on time, temperature, pH, divalent cations and was reversible on addition of excess oCRH. Scatchard analysis revealed a high afinity binding site with a dissociation constant of approximately 0.7 nmol/L and maximum number of binding sites approximately 44 fmol/mg protein. Disuccinimidyl suberate, a chemical cross-linker, was used to covalently attach 125I-oCRH to placental membranes. The labelled placental membranes were analyzed by SDS-PAGE and autoradiography. A major radioactively labelled band with a molecular weight of 55,000 Da was identified. In this study we have identified placental binding sites for CRH with properties similar to CRH receptors described in a number of human and animal tissues and with a molecular weight similar to that of the brain CRH receptor. These binding sites may be involved in the regulation of the placental CRH/ACTH-beta-endorphin axis during pregnancy and parturition.

Correlation of some biochemical parameters with clinical features of protein energy malnutrition.

The authors report on a study correlating the laboratory findings of children with protein energy malnutrition (PEM) with their clinical presentations. Forty-nine cases of marasmus, 17 of kwashiorkor and 28 of marasmic-kwashiorkor, as well as 20 controls, were studied and monitored in hospital. Fasting plasma samples obtained on days 2, 14 and 21 were analyzed for total proteins, albumin, triglycerides and cholesterol. Blood glucose and haemoglobin were also determined. The total plasma proteins and albumin were significantly lower in all the malnourished cases compared to the controls, with marked hypoproteinaemia among those cases with kwashiorkor. Plasma triglycerides were significantly lower in the malnourished group, when compared to controls. With dietary treatment, plasma triglycerides rose significantly accompanied with regression in liver size. Four out of the 17 children with kwashiorkor, had died; they had the lowest serum albumin concentrations of all the cases studied. The mean haemoglobin concentration of those who died was also significantly lower than that of the survivors.

Modulation of the cellular immune response during Plasmodium falciparum infections in sickle cell trait individuals.

Plasma and peripheral blood mononuclear cells (PBMC) were obtained from P. falciparum-infected individuals with and without the sickle cell trait at diagnosis and 7 days after treatment. HbAA and HbAS patients were compared for levels of plasma soluble IL-2 receptors (IL-2R) and the in vitro cellular reactivity to affinity-purified soluble P. falciparum antigens (SPAg), PPD and phytohaemagglutinin (PHA). At diagnosis, HbAS patients with clinical disease had lower plasma-soluble IL-2R levels and parasite counts than the corresponding HbAA patients, whereas HbAS and HbAA patients with asymptomatic infections had comparable soluble IL-2R levels and parasite counts. PBMC from HbAS patients had higher proliferation and IFN-gamma production in response to SPAg than PBMC from HbAA patients. The difference in the lymphoproliferative responses to SPAg between the two groups was evident in patients with asymptomatic infections. In all patients, the clinical severity, the soluble IL-2R levels and the parasite counts were directly related. The former two were inversely related to the proliferative responses to SPAg. After treatment, all the studied parameters were comparable in the two groups. The study indicates that during P. falciparum infection, HbAS compared with HbAA patients had lower in vivo cellular activation and higher in vitro cellular reactivity in response to soluble malaria antigens.

A study of chloroquine and desethylchloroquine plasma levels in patients infected with sensitive and resistant malaria parasites.

This study was carried out on 63 patients in the town of Gadaref in eastern Sudan; each patient was given the standard therapeutic dose of chloroquine (CQ). Plasma levels of chloroquine and its major metabolite desethylchloroquine (DCQ) were measured by means of a high-performance liquid chromatographic method (HPLC) in patients infected with sensitive (sensitive group) and resistant (resistant groups) strains of Plasmodium falciparum. The ratios of chloroquine to desethylchloroquine (CQ/DCQ) in different groups were calculated and the results obtained were compared and correlated with the degree of parasitaemia. The statistical analysis of the results showed that the plasma content of CQ and the CQ/DCQ ratio in the majority of the patients fall within the normal mode of distribution. A small group of patients showed a deviation from the normal mode by having a rather high CQ plasma level and a high ratio of CQ/DCQ. The mean plasma levels of CQ and the CQ/DCQ ratio in the sensitive group was found to be higher than that in the resistant groups. However, these differences were found to be not significant. Correlation tests showed that the levels of CQ and the CQ/DCQ ratios increase with the increase of the degree of parasitaemia in the sensitive group but decrease with the increase of parasitaemia in resistant groups.

Seasonal changes in cell mediated immune responses to soluble Plasmodium falciparum antigens in children with haemoglobin AA and haemoglobin AS.

In this longitudinal study peripheral blood mononuclear cells (PBMC) were obtained before and during the malaria season from healthy HbAA and HbAS children. Cells were compared for proliferation in response to stimulation by soluble Plasmodium falciparum antigens (SPAg) or purified derivative of tuberculin (PPD). The lymphoproliferative responses to SPAg of the paired PBMC samples showed 2 distinct seasonal changes in relation to the haemoglobin phenotype. In HbAA children, the lymphoproliferative responses to SPAg were suppressed during the malaria season. In contrast, they were enhanced in HbAS children during the malaria season. No distinct seasonal change in the response to PPD was found in relation to the haemoglobin phenotype. The study points to the role of the sickle cell trait in modulating the cellular immune responses to falciparum malaria.

Loss of cellular immune reactivity during acute Plasmodium falciparum malaria.

Sixteen patients suffering from acute Plasmodium falciparum malaria were studied. All were residents of an area of unstable malaria-transmission in Eastern Sudan. Blood-samples were drawn at diagnosis, and 7 and 30 days later. Blood-samples from thirteen donors, drawn outside the malaria transmission season 5 months prior to the attack, were included in the study. Lymphoproliferative responsiveness to purified soluble malarial antigens and to the unrelated antigen PPD was lost during the acute phase of the disease in most donors, but was regained during convalescence, except in four donors recrudescing or reinfected by day 30. In contrast to the suppression of antigenic responses, cellular responses to phytohaemagglutinin (PHA) remained virtually unaffected. All donors showed elevated plasma-levels of soluble IL-2 receptor during the acute phase of the disease which normalized during convalescence. Five donors examined by fluorescence-activated cell sorting (FACS) showed no increase in surface expression of IL-2 receptor on peripheral lymphocytes. The data indicate that acute P. falciparum malaria causes a depletion of antigen-reactive T-cells from the peripheral circulation, probably due to homing of this cell-population to lymphoid tissues. It was also found that acute-phase plasma was suppressive to PPD-induced proliferative responses, indicating an additional suppressive mechanism operating in vivo.

Lymphoproliferative responses to Plasmodium falciparum antigens in children with and without the sickle cell trait.

Blood mononuclear cells (BMNC) were isolated from sickle cell trait (HbAS) healthy donors and normal haemoglobin (HbAA) healthy donors resident in a P. falciparum endemic area of eastern Sudan. Blood samples were collected during the malaria season. BMNC were tested for their proliferative responses to phytohaemagglutinin (PHA), purified protein derivative of tuberculin (PPD) and to soluble P. falciparum antigens (SPAg). Higher responses to SPAg and PPD were observed in the HbAS children compared with the HbAA children, whereas no differences were observed among adults of the two phenotypes. Proliferative responses to PHA were comparable in all individuals tested. The significance of these findings in relation to haemoglobin phenotype, age and the possible immunoregulatory mechanisms operating in HbAS and HbAA children during the malaria season is discussed.

The use of plasma fructosamine in the assessment of diabetic control.

Plasma fructosamine and glycosylated haemoglobin were measured in insulin-dependent (n = 30) and in non-insulin-dependent diabetics (n = 90) and in healthy controls (n = 45). Fructosamine correlated significantly with glycosylated haemoglobin in the non-insulin dependent (r = 0.5, P less than 0.001) better than in the insulin-dependent group (r = 0.39, P less than 0.05). No correlation was found between plasma fructosamine and albumin or total protein concentrations when the concentrations of albumin and protein are within normal reference range. The fructosamine test has got many desirable characteristics, which will give it a role in the assessment of diabetic control.

Plasma chloroquine measurement in the evaluation of Plasmodium falciparum sensitivity.

In-vivo assessment of the sensitivity of Plasmodium falciparum to chloroquine was carried out in 63 patients in eastern Sudan. Standard triple-dose therapy with chloroquine (25 mg base kg-1 body wt) failed in curing 30 patients. All grades of resistance were demonstrated in the study, confirming that the phenomenon of chloroquine resistance is well established in this area. Factors which may have accelerated the spread of resistant strains include: a drop in the immunity of the local people caused by previous drought, introduction of non-immune refugees, increased transmission following heavy rains, and massive drug pressure. Plasma levels of chloroquine attained in our patients exceeded the therapeutic level and thus ruled out ineffective levels as a possible cause of treatment failure. Moreover, there was no significant difference between chloroquine levels in patients infected with sensitive or resistant strains. As some patients were cured with an additional dose of chloroquine, it is proposed that chloroquine measurement be carried out in patients treated for falciparum malaria with a view to defining new effective levels for semi-immune populations.

Reduced cellular immune reactivity in healthy individuals during the malaria transmission season.

Antigen-induced cellular immune responses are suppressed during acute malaria. The present study engages the possibility that malaria-induced alterations in cellular immune reactivity extend beyond the clinical disease. Thus, lymphoproliferative responses of healthy individuals were diminished during the malaria transmission period in individuals living in an area of highly seasonal, unstable malaria transmission. This finding may have important implications for the design of studies of stimulatory properties of antigens using lymphocytes of endemic origin.

Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects.

To determine the possible differences in the immune response to Plasmodium falciparum between sickle-cell trait (Hb AS) and normal haemoglobin (Hb AA) individuals, we examined 35 Hb AS and 24 Hb AA subjects matched for age and microenvironment. Their age was 2-55 years and all lived in a malaria endemic area 300 km south of Khartoum. Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) and to circumsporozoite (CS) protein (anti-NANP40) indicated equal exposure to falciparum malaria. Peripheral blood mononuclear cells (BMNCs) from 20/35 (57%) Hb AS subjects compared with 10/24 (42%) Hb AA subjects, responded to affinity-purified P. falciparum soluble antigens (SPAg). Of those responding to SPAg, 9 (26%) Hb AS subjects and only two (8%) Hb AA subjects had high responses. The mean proliferative response to SPAg of BMNCs from Hb AS individuals was significantly higher than in Hb AA individuals (P less than 0.025). Responses of BMNCs to PPD and PHA were also higher among Hb AS individuals and correlated positively with responses to SPAg. These findings support the hypotheses that the sickle-cell trait protects individuals from P. falciparum infections, at least in part, by modulating the immune response.

Cell-mediated immune responses to soluble Plasmodium falciparum antigens in residents from an area of unstable malaria transmission in the Sudan.

This paper describes immune responses to P. falciparum infection in individuals living in an area of highly seasonal, unstable malaria transmission. The in vitro cellular immune responses of peripheral blood mononuclear cells (PBMC) from 36 Sudanese donors to a complex of affinity purified soluble P. falciparum antigens (SPag) and two components thereof (Ag1 and Ag7) were examined and compared to humoral immune parameters. In 29/36 Sudanese donors, SPag induced a significant lymphoproliferative response in vitro. In contrast only 3/27 Danish donors never exposed to malaria responded to SPag. Ag1 and Ag7 induced significant lymphoproliferation in 9/34 and 13/36 Sudanese donors respectively, whereas no Danish donors responded. Significant interferon-gamma production was observed in 16/27, 1/5 and 3/12 Sudanese donors when stimulated by SPag, Ag1 and Ag7 respectively. Lymphoproliferative responses to SPag correlated with proliferative responses to Ag1 and Ag7, and with Spag-induced interferon-gamma production. These results indicate that T-cell clones recognizing epitopes on Ag1 and Ag7 have been expanded in the studied population as a result of exposure to P. falciparum infection. The T-cell parameters did not correlate with the presence of antibodies to SPag, Pf155/RESA or a crude parasite sonicate or with the schizont IFA titers of the plasma. This indicates that parameters outside the degree of exposure to P. falciparum influence the cellular immune responses to malaria.