Plasma haemoglobin determination using chlorpromazine as a non-carcinogenic reagent

Plasma haemoglobin was assayed with the non-carcinogenic reagent phenothiazine. This method is sensitive and allows the measurement of plasma haemoglobin concentrations in the range 4-500 mg/l with a within-run CV of 2.1%, and a between-run CV of 4.3%. A spectrophotometric scanning method (x) based on the determination of haemoglobin as haemiglobin cyanide using the Soret band at 419 nm correlated well with the phenothiazine method (y): y = 1.07x + 15.8, r = 0.995, n = 31. It was found that the absorbances in the phenothiazine method were markedly dependent on the concentration of phosphoric acid.     

Blood haemoglobin determination as haemiglobin isothiocyanate

A method for the determination of blood haemoglobin, as haemiglobin isothiocyanate was developed and evaluated. Appropriate concentrations of the reagents were chosen to minimize the risk of turbidity from plasma proteins. The stability constant of the isothiocyanate derivative was measured, then used to select a KSCN concentration high enough to bring about complete transformation. The method compared fairly well with the standard HiCN method, and with automatic haemoglobin measurements. The proposed procedure has no particular analytical advantages over the standard HiCN method, the haemiglobin azide method, or the alkaline haematin method; but it uses low-toxicity, non-hazardous chemicals.     

The alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood--a candidate reference measurement procedure

The article describes the development and evaluation of the alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood without the need for toxic materials and suitable for use in laboratories in countries with limited resources and restricted import of toxic materials. The validation of the method has been performed in accordance with the requirements set out in the international standard ISO 15193, which describes the procedures necessary for development of a candidate reference measure-ment procedure. The main results were: The trueness of the haemiglobin cyanide (HiCN) method depends upon the diluent used. The international haemiglobin cyanide reference material BCR CRM 522 does not have the same chemical properties as that derived from fresh lysed erythrocytes and cannot be used for calibrating the AHD575 method. The transformation of oxyhaemoglobin to haematin at pH 13 proceeds 5-8 times more rapidly than its conversion to haemiglobin cyanide. The AHD575 method yields results comparable with the HiCN method and uses a readily available crystalline standard of high purity. The introduction of the AHD575 method does not require new reference intervals, the values being directly transferable from (commutable with) the established HiCN procedure.     

Sulfhaemoglobin. Absorption spectrum,millimolar extinction coefficient at λ = 620 nm,and interference with the determination of haemiglobin and of haemiglobincyanide

The spectrophotometric properties of sulfhaemoglobin (SHb) and some derivatives were investigated using an improved technique for measuring the SHb fraction induced in human blood samples. The millimolar extinction co-efficient of SHb at λ = 620 nm was found to be 20.8 (S.D. 1.48; S.E. = 0.44; n = 11). In addition it was demonstrated that the spectral changes occurring in SHb containing haemoglobin solutions upon the addition of KCN, K₃Fe(CN)₆ and K₃Fe(CN)₆ + KCN invalidate the KCN addition method for the determination of haemiglobin. The influence of clinically occurring SHb fractions on the internationally standardized total haemoglobin determination were shown to be insignificant.     

Alkaline haematin D-575, a new tool for the determination of haemoglobin as an alternative to the cyanhaemiglobin method. I. Description of the method

A new method for the rapid and accurate measurement of haemoglobin has been developed as an alternative to the conventional cyanhaemiglobin method. This method is based on the conversion of all haeme, haemoglobin, and haemiglobin species into a stable end product by an alkaline solution of a non-ionic detergent ('AHD reagent'). The reaction product, designated as alkaline haematin D-575, is extremely stable and shows a characteristic absorption peak at 575 nm. As compared to the cyanhaemiglobin method, the determination of haemoglobin by alkaline haematin D-575 offers several advantages such as (1) extreme stability of the AHD reagent and the conversion product, (2) decreased conversion time of all haemoglobin species into the end product, (3) decreased amounts of plasma and cell errors, and errors caused by delayed conversion of carboxy- and fetal haemoglobins, and (4) standardisation by a primary standard (purified crystalline chlorohaemin).     

Haemoglobin A2 measurement using high performance liquid chromatography.

Haemoglobin A2 levels were assessed using a modular high performance liquid chromatography (HPLC) system with a protocol designed for the measurement of haemoglobin A1C. There was good correlation (r = 0.96; P < 0.001) between this technique and the International Committee for Standardisation in Haematology (ICSH) recommended method of microchromatography. The range of haemoglobin A2 was found to be 2.3-3.2% in apparently normal individuals, and 4.0-6.7% in those with beta-thalassaemia trait. The HPLC system produced reliable results quickly for haemoglobin A2 with no alteration to the protocol used for measuring haemoglobin A1C.     

Ethylene glycol-stabilized haemolysates as control material in haemoglobinometry

Human blood haemolysates containing ethylene glycol (final volume fraction 0.35) were prepared and stored at -20 degrees C (in the liquid state) up to 372 days. During the whole period, the total haemoglobin concentration (assayed in the material by means of the reference HiCN method) was found to be stable; spectral analysis also failed in detecting any deterioration, Hi formation being low. Good stability was also recorded on storage at 2-4 degrees C for 15 days, but only for 1-2 days at room temperature. The stabilised haemolysate is suggested as a material for long-term control of accuracy in hemoglobinometry.     

Determination of haemoglobin in gastric aspirates

The haemoglobin content of gastric aspirates can be quantitated by conversion of non-fluorescent haem to fluorescent porphyrins by heating gastric aspirates with oxalic acid and ferrous sulphate. Recovery of haemoglobin added to gastric aspirates was 92 +/- 9%, variation coefficient, n = 52, day to day variation less than 8%. This method was used to calculate blood (haemoglobin) loss in 211 (24 hours) gastric aspirates obtained from 58 intensive care patients. Gastric blood loss was also measured by the 51Cr radiolabelled erythrocytes method in the same samples. There was a good linear correlation (r = 0.942, p less than 0.001) between the two methods. The fluorimetric method of quantitating haem is therefore suitable for detecting and measuring blood loss in gastric contents.     

Measurement of glycosylated haemoglobins using an affinity chromatography method

After removal of the labile material, we have measured the stable glycosylated fraction of haemoglobin with a new, commercially available, phenylboronic acid affinity gel, Glycogel B. The mean value was established for 61 non-diabetics as 7.31 (SD ± 0.92)% and for 108 diabetics as 12.70 (SD ± 2.88)%. The method is highly reproducible with a coefficient of variation below 2.0%. The effect of changing the temperature from 7°C to 37°C, and pH from 8.1 to 8.9 was investigated. For accurate results the temperature should be maintained between 20°C ± 1°C, and the pH between 8.6 ± 0.1. A poor, but significant correlation (r = 0.43) between glycosylated haemoglobin and simultaneous blood glucose was shown. There was a good correlation with the agar gel electrophoretic method (r = 0.95). The slope of the regression Une was 1.20 which indicates that this affinity method measures more than just HbA₁. The affinity method appears to offer greater selectivity for diabetics than the electrophoretic method.     

Serum erythropoietin levels may be inappropriately low in the acute neuropsychiatric porphyrias

BACKGROUND: Patients with the acute porphyrias may develop renal failure and autonomic dysfunction. Renal damage and sympathetic failure may both cause erythropoietin (EPO) deficiency. In this study, we have investigated serum erythropoietin levels and autonomic function in patients with acute porphyria in clinical remission. METHODS: Serum erythropoietin levels and the corresponding haemoglobin (Hb) were assayed in 31 patients with acute porphyria and were compared to 15 type 1 diabetic patients with autonomic neuropathy, 23 patients with iron-deficiency anaemia and 18 healthy individuals. RESULTS: 9 out of 31 porphyric patients showed a normochromic normocytic anaemia with normal ferritin levels. Three patients had borderline-raised serum creatinine levels, and one of them was anaemic. Autonomic function was investigated in seven patients, six of them being anaemic, and the results were normal. Patients with iron-deficiency anaemia showed the expected increase in serum erythropoietin levels in response to a decreasing haemoglobin (r=-0.86, p<0.001). Patients with porphyria had inappropriately low serum erythropoietin levels for the degree of anaemia compared to iron-deficiency patients (p<0.001) although there was still a significant increase in serum erythropoietin with decreasing haemoglobin levels (r=-0.46, p=0.01). In contrast, diabetic autonomic neuropathy patients demonstrated a significant decrease in serum erythropoietin with decreasing Hb levels (r=+0.53, p=0.05). CONCLUSIONS: Patients with acute porphyria may have inappropriately low levels of EPO. In contrast to the diabetic patients, this does not appear to be due to autonomic neuropathy but it may reflect mild renal tubular impairment.     

Use of strain imaging in detecting segmental dysfunction in patients with hypertrophic cardiomyopathy.

BACKGROUND: The distribution and magnitude of left ventricular hypertrophy are not uniform in patients with hypertrophic cardiomyopathy (HCM). Previous echocardiographic studies have focused on global left ventricular function. Recently, myocardial Doppler strain (epsilon) imaging, a newly developed technique, has allowed the quantification of regional myocardial motion. The aim of this study was to characterize regional left ventricular systolic function by myocardial Doppler epsilon imaging in patients with HCM. METHODS: Included in this study were 31 patients with asymmetric septal hypertrophy and HCM, and 41 age-matched healthy patients. Regional longitudinal axial systolic epsilon was assessed at the basal, mid, and apical segments of the septal and lateral walls and compared between both groups. RESULTS: Patients with HCM had reduced epsilon at the ventricular septum (-10.3 +/- 5.7%) compared with control patients (-19.4 +/- 3.3%, P <.001). In the HCM group, epsilon in the midseptum (-1.3 +/- 8.2%) was significantly less than at the basal (-12.2 +/- 8.7%, P <.01) and apical septum (-17.3 +/- 10.4%, P <.01), and was also less than at the midlateral wall (-9.4 +/- 5.3%, P <.05). There was a significant correlation between midseptal epsilon and intraventricular septum to posterior wall thickness ratio (r = 0.81, P <.001). CONCLUSION: Midseptal longitudinal epsilon was markedly decreased, even reversed in patients with HCM (paradoxic longitudinal systolic expansion), which was directly related to the degree of septal hypertrophy. Myocardial Doppler epsilon imaging could offer a unique approach to quantify regional systolic dysfunction in these patients.     

Remnant-like particle cholesterol levels in Korean patients with coronary artery disease and non-insulin dependent diabetes mellitus.

Several studies have provided evidence that the remnants of lipoproteins may be the atherogenic components of triglyceride-rich lipoproteins. The purpose of this study was to investigate whether the remnant-like particle cholesterol (RLP-C) is an independent risk factor for coronary artery disease (CAD) and non-insulin dependent diabetes mellitus (NIDDM) in the Korean population and to explore the relationship between RLP-C and other biochemical markers as well as the apolipoprotein (apo) E genotypes. Lipid and lipoproteins including RLP-C and apo E genotypes were analyzed in 98 normal adults (control group), 68 patients with CAD (CAD group), 88 patients with NIDDM (DM group), and 19 patients with both CAD and NDDM (CAD + DM group). RLP-C levels were significantly higher in the DM (p < 0.0001), CAD (p = 0.0012) and the CAD + DM groups (p = 0.0184) than in the controls. To determine which variable could discriminate most effectively and independently among the different groups, stepwise linear discriminant analysis was performed for all the variables that showed p < 0.15 by univariate analysis. RLP-C was selected as an independent discriminator between the control and patient groups. RLP-C levels showed a strong positive correlation with trigylceride levels in the control, CAD and DM groups (r = 0.783, r = 0.610 and r = 0.746, respectively). In overall groups, apo epsilon4 and epsilon2 carrier genotypes showed a significant increase in RLP-C levels compared with epsilon3/3 wild-type (p = 0.0085). After adjusting for the effect of apo E genotypes, a significant increase of the RLP-C levels in the disease groups remained. In conclusion, RLP-C was determined to be an independent risk factor in Korean patients with CAD and NIDDM and showed a strong correlation with triglyceride levels. We suggest that the increased cardiovascular risk associated with the epsilon4 and epsilon2 allele may be mediated by more atherogenic RLP-C.     

The relative extent of glycation of haemoglobin and albumin

The level of non-enzymatic glycation of a protein is thought to depend on the number of sites available for reaction, the half-life of the protein and the ambient concentration of glucose. Accordingly, the modification of two blood proteins with a similar number of potential sites but different survival times was examined in non-diabetic patients by periodate oxidation and by reduction with [3H]borohydride. The amount of glycation of haemoglobin and its sub-fractions HbA1 and HbA1c were determined to be 0.44, 2.42 and 2.24 mol/mol respectively and the corresponding value for albumin was 0.37 mol/mol protein. Amino acid analysis showed that the epsilon amino groups of albumin were more extensively modified than they were in haemoglobin and thus it is concluded that the average rate of reaction of the lysine residues in albumin is markedly faster than in haemoglobin.     

Impact of parturition on iron status in nonanaemic iron deficiency

BACKGROUND: Iron-deficient nonanaemic parturients risk underdiagnosis as a result of the reliance on postpartum ferritin and haemoglobin as markers of iron status. Ferritin is an acute-phase protein whose levels increase during the inflammatory response, as occurs after delivery. Our aims were to evaluate the impact of parturition on iron status, erythropoiesis and the inflammatory response, and identify the optimal parameters and timing for diagnosing iron deficiency in the presence of postpartum inflammation. MATERIALS AND METHODS: Conventional parameters of iron status, erythropoiesis and the inflammatory response (serum ferritin, serum iron, transferrin saturation, C-reactive protein) were compared with more recent parameters [soluble transferrin receptors (sTfR), hypochromic red cells, reticulocyte indices] within 48 h either side of delivery in 64 iron-deficient nonanaemic women (defined by a prepartum serum ferritin < or =15 microg L(-1), and a pre- and postpartum haemoglobin of > or =11.0 g dL(-1) and > or =10.0 g dL(-1), respectively). RESULTS: Mean sTfR decreased pre to postpartum from 7.3 to 5.8 microg mL(-1) (P<0.01), while mean serum ferritin increased from 9.7 to 16.9 microg L(-1) (P<0.01). Serum ferritin did not correlate with haemoglobin pre or postpartum (r=0.04, P=0.7; r=0.2, P=0.09), but a correlation persisted postpartum between hypochromic red blood cells and haemoglobin (r=-0.26; P<0.05). The percentage of hypochromic red cells remained virtually unchanged pre- and postpartum (4.0% vs. 3.8%; NS). Postpartum mean reticulocyte haemoglobin content (CHr) was 27.1 +/- 1.6 pg. CONCLUSION: Iron status should be tested prepartum, in the absence of an inflammatory response, rather than in the early postpartum. A valuable additional parameter, where available, might be the hypochromic red cell percentage, which is virtually uninfluenced by the inflammatory response. Furthermore, hypochromic red cell percentage, CHr and sTfR can be helpful to differentiate between functional iron deficiency and depleted iron stores.     

Apolipoprotein E gene polymorphism frequencies in a sample of healthy Hungarians.

Apolipoprotein E (apo E) has been found to play an important role in lipid metabolism and has been associated with cardiovascular and neurodegenerative conditions. Hungarians have some of the highest rates of cardiovascular morbidity and mortality in the world. This study examines the distribution of apo E alleles and genotypes in a population of healthy ethnic Hungarian blood donors (n = 302). Male (n = 152) and female (n = 150) subjects ranging from 18 to 62 years of age (mean 37.0) were involved. To determine the frequency of apo E alleles, polymerase chain reaction followed by restriction length polymorphism was applied. The analyses of data showed that apo E allele epsilon3 had the greatest frequency in this group (0.807), followed by apo epsilon2 (0.104) and apo epsilon4 (0.087). The highest genotype frequency was found to be epsilon3/3 at 65.2% (n = 197) followed by genotype epsilon3/4 at 15.9% (n = 48), genotype epsilon2/3 at 15.2% (n = 46), genotype epsilon2/2 at 2.3% (n = 7), genotype epsilon2/4 at 1.0% (n = 3) and genotype epsilon4/4 at 0.4% (n = 1). The apo E frequencies found in this study appear to differ from an earlier study of blood donors, where the results are based on apo E phenotyping.     

Headache prevalence related to haemoglobin and ferritin. The HUNT Study

As clinic-based studies show an association between headache and both high and low levels of haemoglobin, we analysed this relationship in a population-based cross-sectional study (the HUNT Study). A total of 2385 women aged 20-55 years responded to a headache questionnaire and gave blood samples for measuring haemoglobin and ferritin. In the multivariate analyses, adjusting for age and education, there was a linear trend of decreasing prevalence of headache (P = 0.02) and migraine (P = 0.01) with decreasing haemoglobin. In particular, migraine was less likely among women with low haemoglobin (values < 11.5 g/dl) (odds ratio 0.4, confidence interval 0.2, 0.9). There was no correlation between headache prevalence and ferritin. The present findings may be relevant for the headache reported in polycythaemia and chronic altitude sickness.     

The validity of oxygen content calculations

Blood oxygen content calculated from haemoglobin concentration, measured haemoglobin oxygen saturation and measured oxygen tension was compared with three other methods of estimating oxygen content. These other methods were those of Van Slyke and Zander, which are direct methods, and a method using Kelman's equation to estimate the saturation from meassured oxygen tension and hence content. The coefficients of correlation (corr coeff) (r) were 0.9050 (n = 22), 0.9919 (n = 24) and 0.9862 (n = 25) for the respective methods when compared with oxygen content calculated using measured saturation. The Van Slyke method proved to be imprecise in our hands. The direct measurement using the oxygen cuvette of Zander gave oxygen content values similar to those estimated from measured saturation. The oxygen content calculated from pO₂ alone when compared to that derived from measured saturation had a corr coeff (r) of 0.9862 (n = 25), but the high residual standard deviation (So) of 6.939 ml/l indicates that the practice of calculating oxygen content from oxygen tension alone is imprecise. We conclude that oxygen content may be satisfactorily estimated by the Zander method when it becomes generally available, but until then the measurement of oxygen saturation is a necessary prerequisite to the estimation of blood oxygen content.     

One-dimensional ultrasonic strain and strain rate imaging: a new approach to the quantitation of regional myocardial function in patients with aortic stenosis

Abnormalities in regional left ventricular (LV) function in aortic stenosis (AS) have yet to be appropriately characterized. One-dimensional strain (epsilon) and strain rate imaging (SRI), new ultrasound (US) indices for quantifying regional wall deformation, might allow this. The aims of this study were 1. to define regional radial and longitudinal epsilon /SR in AS; 2. to establish if they are related to the severity of the disease; and 3. to determine if regional deformation is further altered by coexistent coronary artery disease (CAD). A total of 40 patients were studied: Group I with isolated AS (10 women, 10 men; mean age 66 years) and group II with AS and concomitant CAD (CAD/AS) (13 women, 7 men, mean age 68 years). Data were compared to 20 age-matched healthy people (N). Regional systolic maximal velocity/SR and end-systolic and maximal epsilon were measured. The maximal systolic velocity/SR in AS and CAD/AS patients were significantly reduced compared to N. The two patient groups could be further differentiated by end-systolic and maximal epsilon, which demonstrated a further reduction in both epsilon indices in CAD/AS (i.e., maximal radial epsilon 29.3%, AS; 23.7%, CAD/AS; 40.4%, N; AS and CAD/AS vs. N, AS vs. CAD/AS, p < 0.05). Indices of radial and longitudinal deformation correlated both with aortic valve area (AVA) and stroke volume (SV) (i.e., radial maximal epsilon and AVA, r = 0.77, p < 0.05). A significant correlation was also found between epsilon indices and the severity of left anterior descending (LAD) or circumflex artery (CX) coronary artery. Regional myocardial deformation in AS is abnormal. In the absence of CAD, the degree of abnormality correlates with aortic valve area (AVA). The severity of the disease was best expressed by changes in regional epsilon. In CAD/AS patients, there was a significant further reduction in end-systolic and maximal epsilon. These changes correlated with the severity of coronary narrowing in the subtending vessel.     

APOE polymorphism and the progression of diabetic nephropathy in Japanese subjects with type 2 diabetes: results of a prospective observational follow-up study

OBJECTIVE: The aim of this study is to clarify the conflicting results of the epsilon2/epsilon3/epsilon4 APOE polymorphism as a risk factor on diabetic nephropathy by a cohort study. RESEARCH DESIGN AND METHODS: A total of 429 Japanese subjects with type 2 diabetes and with normoalbuminuria (n = 299) or with microalbuminuria (n = 130) were enrolled in a prospective observational follow-up study during 1995-1998 and followed until 2001 (for at least 3 years). The endpoint was the occurrence of a renal event defined as the progression to a higher stage of diabetic nephropathy. RESULTS: During the study (the mean follow-up period: 4.4 +/- 1.0 years), 31 of 429 subjects progressed: 21 from normoalbuminuria to microalbuminuria and 10 from microalbuminuria to overt proteinuria. The allele frequency of the APOE polymorphism was significantly different between the progressors and the nonprogressors. Eight of 42 epsilon2 carriers (19%) progressed, whereas 23 of 387 noncarriers (6%) progressed with a relative risk of 3.2 (95% CI 1.5-6.7). When subjects were stratified by renal status at baseline, each relative risk for the progression in the epsilon2 carriers was 2.7 (0.99-7.4) in those with normoalbuminuria and 4.2 (1.3-13.3) in those with microalbuminuria. Furthermore, when analyzed only in subjects with normoalbuminuria and short duration of diabetes (<15 years) at baseline, the risk in the epsilon2 carriers became higher to 3.2 (1.2-8.8). CONCLUSIONS: Our follow-up study indicates that the epsilon2 allele of the APOE polymorphism is a prognostic risk factor for both the onset and the progression of diabetic nephropathy in Japanese type 2 diabetes.     

Determinants of intra-individual variation in kidney function in normoalbuminuric insulin-dependent diabetic patients: importance of atrial natriuretic peptide and glycaemic control.

1. In order to investigate the modulation of kidney function in insulin-dependent diabetes mellitus, intraindividual variation in glomerular filtration rate, renal plasma flow, urinary albumin excretion rate and mean arterial blood pressure was assessed in 22 normoalbuminuric patients [age 31 +/- 8 years, duration of diabetes 9 +/- 5 years, mean arterial blood pressure 90 +/- 5 mmHg (means +/- SD), urinary albumin excretion rate 5.4 x/divided by 1.6 micrograms/min]. The variation in these parameters was calculated from the results of two clearance studies (continuous infusion of [125I]-iothalamate and 131I-hippuran as markers for glomerular filtration rate and renal plasma flow, respectively) and was subsequently analysed in relation to individual variation in plasma concentrations of atrial natriuretic peptide, arginine vasopressin, angiotensin II and aldosterone and measures of glycaemic control. 2. Simple correlation analysis showed a significant association between intra-individual variation in glomerular filtration rate and atrial natriuretic peptide (sigma = 0.66, P = 0.003). Besides variation in atrial natriuretic peptide, multiple regression analysis identified variation in glycated haemoglobin (P = 0.026) and arginine vasopressin (P = 0.057) as variables having independent association with variation in glomerular filtration rate [R2 with the three variables included (adjusted for degrees of freedom) = 0.50, analysis of variance: P = 0.002]. 3. With respect to variation in renal plasma flow, differences in fasting blood glucose concentration and mean arterial blood pressure were suggested as determinants (R2 = 0.36, analysis of variance: P = 0.009). 4. Variation in urinary albumin excretion rate (after log transformation) was statistically associated with variation in glycated haemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)