Frank Niklasson

A systematic study of nucleotide analysis of human erythrocytes using an anionic exchanger and HPLC

A two-step procedure for rapid HPLC analysis of nucleotides from human erythrocytes is described. A strong anion exchanger Partisil-10 is used as column material. Elution of monophosphates requires about 12 min and the elution of di- and triphosphates 21 min. The elutions are performed separately and with different injections as such a procedure will save time otherwise used to re-condition the columns. In addition to the three adenylates other nucleotides such as GTP, GDP, IMP and NAD can be recorded as isolated, well-defined peaks, which can be subject to quantitative analysis. The mean value of ATP concentration for 12 healthy individuals was 1.55 +/- 0.05 mmol/l, about 25% higher than generally reported in the literature. Accurate estimates of ADP and AMP concentrations allowed calculation of mean values for the physiologically interesting ratios: adenylate energy charge (0.945 +/- 0.002) and equilibrium constant for adenylate kinase (1.205 +/- 0.053). GDP and GTP are present in concentrations that are about 4% of those for corresponding adenylates. The analysis of nucleotides in human erythrocytes is a useful way of studying erythrocyte preservation and investigating patients with hemolytic disorders.

Plasma concentration and renal excretion of adenine and 2,8-dihydroxyadenine after administration of adenine in man

A new method of high performance liquid chromatography (HPLC) which makes it possible to analyse 2,8-dihydroxyadenine (DOA) in plasma in concentrations exceeding 0.25 mumol/l is described. The method was used to study the renal elimination of DOA. For comparison, the renal handling of adenine was also investigated. The results from an analysis of the experimental data support the assumption that more than one concentration-dependent mechanism exists in the renal tubuli for each of the two purines, adenine and DOA. In general the clearance values are higher for DOA than for adenine and indicate net secretion for both substances.

Population-based pediatric reference intervals for hematology, iron and transferrin

Abstract Reference intervals are crucial decision-making tools aiding clinicians in differentiating between healthy and diseased populations. However, for children such values often are lacking or incomplete. Blood samples were obtained from 689 healthy children, aged 6 months to 18 years, recruited in day care centers and schools. Hematology and anemia analytes were measured on the Siemens Advia 2120 and Abbott Architect ci8200 platforms (hemoglobin, erythrocyte volume fraction [EVF], erythrocytes, mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], reticulocytes, leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, basophils, platelets, iron, transferrin, transferrin saturation). Age- and gender-specific pediatric reference intervals were defined by calculating 2.5th and 97.5th percentiles. The data generated is primarily applicable to a Caucasian population, but could be used by any laboratory if verified for the local patient population.

Amino acid concentrations in cerebrospinal fluid in presenile and senile dementia of Alzheimer type and multi-infarct dementia

Free amino acid levels were measured in cerebrospinal fluid (CSF) from demented patients (D, n = 30) suffering from presenile and senile dementia of Alzheimer type (PDAT, n = 7; SDAT, n = 9), multi-infarct dementia (MID, n = 14) and a reference sample group consisting of young neurotic patients (R, n = 16). Comparing the amino acid levels in the dementia subgroups, significantly higher alanine, methionine, phenylalanine and tyrosine levels were found both in MID and SDAT vs. PDAT. No difference was seen between SDAT and MID. Compared to the reference sample group, higher glycine levels were found in each dementia subgroup; higher alanine, methionine and ornithine levels in MID, and SDAT; and higher phenylalanine levels in MID. In PDAT the level of tyrosine was lower. Coefficients of correlation were calculated between amino acid levels and age, and the findings in the reference sample groups were divergent from those observed in dementia. The differences observed are discussed in terms of amino acid, carbohydrate and neurotransmitter metabolism.

Reference intervals on the Abbot Architect for serum thyroid hormones, lipids and prolactin in healthy children in a population-based study

Abstract Pediatric reference intervals for thyroid hormones, prolactin and lipids are of high clinical importance as deviations might indicate diseases with serious consequences. In general, previous reference intervals are hampered by the inclusion of only hospital-based populations of children and adolescents. The study included 694 children, evenly distributed from 6 months to 18 years of age. They were recruited as volunteers at child care units and schools. All subjects were apparently healthy and a questionnaire on diseases and medications was filled out by parents and by the older children. TSH, free T4, free T3, total cholesterol, LDL, HDL, triglycerides and prolactin were analyzed on Abbott Architect ci8200. Age- and gender-related 2.5 and 97.5 percentiles were estimated. The thyroid hormone levels were similar to previous data for the Abbott Architect platform, but exhibited differences from studies performed with other methods. Prolactin displayed wide reference ranges, but relatively small age-related changes, and a marginal difference between sexes during adolescence. Reference intervals for lipids in the different age groups are known to vary geographically. Levels of LDL and total cholesterol were higher than those reported for children in Canada, but lower than those reported for children in China. The study gives age- and gender- specific pediatric reference intervals, measured with modern methods for a number of important analytes. The results presented here differ from previously recommended reference intervals. In many earlier studies, retrospective hospital-based reference intervals, which may include various sub-groups have been presented. By non-hospital studies it is possible to avoid some of these biases.

Population-based pediatric reference intervals for HbA1c, bilirubin, albumin, CRP, myoglobin and serum enzymes.

Abstract Background. Many previous studies on reference intervals are hampered by the inclusion of only hospital-based populations of children and adolescents. Methods. This study included 694 children, evenly distributed from 6 months to 18 years of age. They were recruited as volunteers at child care units and schools. All subjects were apparently healthy. A questionnaire on diseases and medications was filled out by parents and by the older children. Results. Alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), bilirubin, conjugated bilirubin, C-reactive protein (CRP), creatine kinase (CK), Gamma-glutamyltransferase (GGT), HbA1c (mono S and IFCC calibrations), lactate dehydrogenase (LD), myoglobin and panceratic amylase were analyzed on Abbott Architect ci8200, and for HbA1c on Tosoh G7 and a mono S-system. Age- and gender-related 2.5th and 97.5th percentiles were estimated. For some analytes the differences to comparable studies were substantial. Conclusion. The study gives age- and gender-specific pediatric reference intervals, measured with modern methods for a number of important analytes. The results emphasize the importance to evaluate pediatric reference intervals in different populations and ethnic groups including only healthy subjects.

Purine metabolites in the CSF in presenile and senile dementia of Alzheimer type, and in multi infarct dementia

Concentrations of hypoxanthine, xanthine, uric acid and creatinine were measured in CSF of patients suffering form presenile and senile dementia of Alzheimer type (PDAT, SDAT) and multi infarct dementia (MID) and in a reference group of young neurotic patients. There was no difference in hypoxanthine concentration, but there was a marked elevation of xanthine concentration in each dementia group, independent of the type of dementia. There was a significant elevation of uric acid in SDAT and MID but not in PDAT. The concentration of uric acid was higher in MID than in SDAT. There was a higher level of creatinine in the dementia groups, but no difference was seen among the dementia groups. These results are discussed in order to better interpret the etiology and the differentiated diagnosis of the types of dementia.

Purine metabolism in normal and ITP-pyrophosphohydrolase-deficient human erythrocytes

Fresh and stored erythrocytes from normal and ITP-pyrophosphohydrolase (ITP-ase, EC 3.6.1.19) deficient individuals were incubated with hypoxanthine, guanine, allopurinol, and inosine. Differences in the purine metabolism between the normal and the ITP-ase deficient erythrocytes were observed only in the IMP-ITP cycle. Hypoxanthine, guanine and allopurinol were converted to nucleotides at the same rate. Hypoxanthine (2.5 mumol/l) inhibited the salvage of allopurinol (40 mumol/l). A slow decrease (0.7%/day) in salvage rate was observed in both types of cells upon storage at +4 degrees C. Erythrocyte ITP-ase activity was measured in a reference sample group of 48 healthy volunteers. Two distinct groups were found with mean activities equal to 48.3 +/- 13.1 nkat/g Hb (means +/- SD, n = 38) and 11.4 +/- 4.3 nkat/g Hb (n = 10). In two previously selected subjects, the ITP-ase activity was 0.2 and 2.4 nkat/g Hb. A hypothetical genetic mechanism is discussed. The maximal energy turnover in the IMP-ITP cycle during hypoxanthine incubation was found to be less than 10% of the basal erythrocyte energy turnover.

Adenine metabolism in man 1. After intravenous and peroral administration

The basal plasma concentration of adenine and its renal excretion was studied in two men. For its analysis partly new chromatographic techniques were developed. The plasma concentration varied around 70 nmol/l; the renal excretion rate was, as reported earlier by other investigators, around 10 nmol/min. Loadings, intravenously during about 3 h and orally, both with about 30 nmol adenine per kg body mass revealed that most of the adenine was metabolized to nucleotide form. In the experiments with intravenous administration of adenine only about 2% of the given dose appeared in the urine as adenine and somewhat less as the easily precipitable metabolite 2,8-dihydroxyadenine. In the peroral loads, with higher plasma adenine concentrations, the ratios of the renally excreted two compounds were one to a few per cent higher.

Allopurinol kinetics in humans as a means to assess liver function: Evaluation of an allopurinol loading test

A newly developed liver function test was performed on 18 apparently healthy individuals and 29 patients with liver disease. After intravenous injection of a low dose allopurinol (17.1 mumol/kg body mass), blood specimens were collected during 1 h. Plasma analyses of allopurinol and its metabolite oxipurinol were performed and the data were processed by means of a computer-based biodynamic model. This modelling approach makes it possible to estimate parameters, containing information about liver perfusion, hepatocyte membrane transport and hepatocyte cell mass. One parameter (kA31) showed complete discrimination between the reference sample group of healthy individuals and patients with severe liver dysfunction. In a reference sample group of patients with slightly to moderately reduced liver function, only a few patients (5/20) had a kA31 value over the decision limit. In this respect, the allopurinol loading test is superior to the conventional intravenous galactose tolerance test.