Siegbert Bissbort

Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum.

A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiolspecific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory.

Ethnic immunity to coronary heart disease

Dietary fat intake is often regarded as a major determinant of coronary heart disease (CHD) rate and it has been deemed unnecessary to invoke racial or other factors to explain the differences in CHD rates among different ethnic groups. Despite a high prevalence of CHD risk factors such as hypertension, obesity, and smoking, CHD remains a rarity in westernized black Africans. Cord blood total cholesterol (TC), low density lipoprotein cholesterol (LDLC) and apolipoprotein B (apo B) levels were measured and found to be respectively 12.1%, 18.3% and 22.4% lower in black neonates when compared to white neonates. These differences were again studied in a group of young black African males and a comparable group of age-matched whites who had been exposed to the same environment and western diet for at least 2 years. Although the body mass indices and serum albumin concentrations in the adult males were not significantly different, serum levels of TC, LDLC and apo B were 10.7%, 18.7% and 39.7% lower in the blacks, respectively. Furthermore, high density lipoprotein cholesterol (HDLC) and Apolipoprotein AI were 20.2% and 9.5% higher, homocysteine 45.6% lower and coagulation factor VII 26.6% lower in the adult black Africans. It is concluded that blacks are biochemically less responsive to an atherogenic diet than whites and these differences are already present at birth.

Novel test and its automation for the determination of erythrocyte acetylcholinesterase and its application to organophosphate exposure

BACKGROUND Because of the lack of a problem-free, reliable method for determination of erythrocyte acetylcholinesterase (AChE), we developed a simple kinetic method, which we found to be both reliable and suitable for automation in the routine clinical laboratory. METHODS Acetylthiocholine, used as substrate, is hydrolysed by acetylcholinesterase to yield acetate and thiocholine. Thiocholine reacts with dichlorophenolindophenol, a blue coloured compound, which is reduced to a colourless product, producing a linear decrease in absorption at 606 nm. If required, this assay can also be run at 600 nm with equally acceptable results. RESULTS The method was automated on the Synchron LX20 multianalyser (Beckman Instruments) and blood samples of 80 patients with clinically symptomatic organophosphate poisoning and 153 normal controls were evaluated. Acetylcholinesterase values were in the range of 0-14 UgHb(-1) in cases of organophosphate poisoning, in contrast with normal controls, who had AChE values of 24.4--37.9 UgHb(-1). No overlap was found between AChE values of controls and poisoned cases. Intra- and inter-assay coefficients of variation were 1.68 and 3.71%, respectively. CONCLUSION The method we propose for measurement of AChE was found to be simple, reliable and easily automatable in the routine clinical laboratory.

Adenosine deaminase isoenzymes in typhoid fever.

The activity of serum adenosine deaminase and its isoenzymes (ADA1 and ADA2) was measured in the sera of 55 patients with clinically diagnosed typhoid fever, 46 of whom were seropositive forSalmonella typhi infection. At presentation 95% of the serologically positive patients had increased serum adenosine deaminase activity. The increase in activity was greater than that of other frequently measured enzymes. Isoenzyme ADA2 accounted for 84% of the activity, indicating a monocyte/macrophage origin. Results show that in patients with typhoid fever, serum adenosine deaminase, particularly its isoenzyme ADA2, may be regarded as a marker for monocyte/macrophage activation and could be valuable in diagnosis.

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (+/- S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B6-deficient men was 5.04 +/- 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p = 0.005) lower than the 6.69 +/- 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B6 status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B6 deficiency.

An enzymatic assay of inorganic phosphate in serum using nucleoside phosphorylase and xanthine oxidase

We have developed a new enzymatic assay for the determination of inorganic phosphate (Pi) in serum, using nucleoside phosphorylase (NP) and xanthine oxidase (XOD). Pi and inosine react to form hypoxanthine and ribose-1-phosphate. The hypoxanthine is oxidized to xanthine, which is further oxidized to uric acid. In these two reactions 2,6-dichlorophenol-indophenol (DCIP) is reduced to a colourless compound and the decrease in colour is measured spectrophotometrically at 600 nm. The assay is automated with an RA-XT analyser. The precision of the automated assay is acceptable (C.V. < 3.5%) and results are accurate and linear across a range of values from 0.2-2.5 mmol/l. The assay correlates well with molybdate methods carried out on SMAC III and RA-XT analysers (r values 0.99 and 0.98, respectively), and seems to be less prone to non-specific sample interference than the usual RA-XT method. The enzymatic assay described seems to be suitable for the routine determination of serum Pi.

Chicken eggshell porphyrins and the glyoxalase pathway: its possible physiological role.

1. The plasma 4,5-dioxovaleric acid (DOVA) levels of two different breeds of chicken were determined and found to be higher in the group with a higher porphyrin eggshell content. 2. The erythrocyte and uterus glyoxalase II activity, investigated by means of a new spectrophotometric method, was found to be significantly higher in the group with a low porphyrin eggshell content. 3. A comparative genetic study of two chicken populations, one with white and one with dark eggshells, showed different gene frequencies for the glyoxalase I polymorphism. 4. An interpretation of these data suggests that the glyoxalase pathway may be involved in the metabolism of early porphyrin precursors.