Assessment of vitamin A status of preschool children in a sub-Saharan African setting: comparative advantage of modified relative-dose response test.

A nationally-representative sample of 2,696 preschool children living in Congo was examined during Au gust-September 2003 to determine the rates of vitamin A deficiency. Ninety clusters of 30 children, aged six months to six years, were selected, using a randomized two-level cluster-sampling method. Vitamin A deficiency was determined by assessing the prevalence of active xerophthalmia (nightblindness and/or Bitot spots) in the cross-over sample of 2,696 individuals. A semi-quantitative seven-day dietary questionnaire was concurrently applied to the mothers of children enrolled to estimate the latter's consumption of vitamin A-rich food. Vitamin A status was assessed by performing the modified relative dose-response test (MRDR) on dried blood spots (DBS) from a subsample of 207 children aged less than six years and the impression cytology with transfer (ICT) test on a subsample of 1,162 children. Of the children enrolled, 5.2% suffered from nightblindness, 8.0% had Bitot spots, and 2.5% had other vitamin A deficiency sequellae. Fifty-three percent of the ICT tests showed the presence of vitamin A deficiency. The biochemical MRDR test showed that the vitamin A status of 30% of the study children was critical. Twenty-seven of them had retinol levels of < 10 microg/dL [mean +/- standard deviation (SD) 7.02 +/- 2.0 microg/dL], and 50% had retinol levels of 10-20 microg/dL (mean +/- SD 14.2 +/- 2.83 microg/dL). The poor health status and low rates of consumption of vitamin A-rich food are the main factors determining critical status. Vitamin A deficiency, reflecting poor nutrition and health, is a serious public-health issue among children aged less than six years in Congo.

Prevalence of infant Vitamin A deficiency and undernutrition in the Republic of Congo.

OBJECTIVES: A representative sample of 5722 pre-school children living in rural and urban areas of the Congo was examined between July and September 1999 for assessing Vitamin A deficiency. METHODS: Using a randomized two-level cluster sampling method, 190 clusters of 30 children aged from 6 months to 6 years were selected in order to assess the prevalence of active xerophthalmia (night blindness and/or Bitot spots). Concurrently, the children's height and weight were determined. A semi-quantitative seven-day dietary questionnaire was applied to the mothers of 5722 children to estimate the latter's consumption of Vitamin A rich foodstuffs. The prevalence of biochemical deficiency was assessed based on the serum retinol concentrations analyzed in dried blood spots from a sub-sample of 300 children living in the Pointe-Noire area. RESULTS: Among the 5722 children studied, 0.7% were found to suffer from night blindness and 7.7% had Bitot spots. The weekly intake of Vitamin A rich foods was estimated in 5722 children. Our data suggest that Vitamin A rich food consumption was lower in rural zones than in urban area according to the food frequency method threshold values. The serum retinol levels were lower than 10 microg/dl in 18% (95% confidence interval [C.I.]: 13.7, 22.3) [8.04+/-2.87 microg/dl] and less than 20 microg/dl in 49% (95% C.I.: 43.4, 54.6) [15.05+/-2.76 microg/dl] of the 300 studied children. We have established a significant relation between mean serum retinol levels and high rate of Vitamin A food intake (chi-square=59.64, 2 d.d.l., p<0.05) in the sample studied. The mean serum retinol concentrations did not differ significantly between the various Z-scores of weight for age (W/A) and height for age (H/A) patterns. But children with a weight for height (W/H) ratio below -2 standard deviation (S.D.) had significantly lower serum retinol values [9.33+/-1.3 microg/dl] than those with a W/H ratio greater than or equal to -2S.D. [10.82+/-4.84 microg/dl]. CONCLUSION: These data suggest that Vitamin A deficiency is still a serious public health problem in rural areas of the Congo in which this study was carried out.

Quantification of urinary allantoin by capillary zone electrophoresis during recombinant urate oxydase (rasburicase) therapy.

OBJECTIVES: Rasburicase (Fasturtec) is used to prevent or treat hyperuricemia associated with chemotherapy. We developed a capillary zone electrophoresis method to measure urinary allantoin, the degradation product of uric acid by rasburicase. DESIGN AND METHODS: Electrophoresis was performed using a P/ACE 5500 system (Beckman) with a fused silica capillary tube and a UV-visible detector set at 214 nm. Urine samples from 10 patients with non-Hodgkin's lymphoma were analyzed to validate the technique. RESULTS: Using a sodium tetraborate running buffer, urinary allantoin was separated from related compounds and internal standard in less than 30 min. The method was linear up to 1.25 g/L (quantification limit: 30 mg/L); precision was below 10%. The total amount of allantoin excreted in patients treated by rasburicase ranged from 1.5 g to 7.9 g/4 days. CONCLUSION: This CZE assay is a simple, rapid and reproducible method to measure allantoin in urine. Different elimination profiles have been found in patients treated with rasburicase.

[Dried blood spot Vitamin A determination by high pressure liquid chromatography with electrochemical detection]. / Dosage de la vitamine A sur taches de sang séché par chromatographie liquide haute pression avec détection électrochimique.

In tropical countries. vitamin A deficiency is one of the most important dietary deficiencies. Its monitoring usually involves analysis of retinol after venipuncture with some difficulties (disease transmission, religious belief). Sample collection on Dried Blood Spot (DBS) is less invasive and safer. Sample storage is easier. We developed a liquid chromatography method with electrochemical detection to measure DBS retinol. Retinol acetate was used as an internal standard. The method is linear up to 2.5 microM with a detection limit of 0.04 microM. Precision is below 10% and DBS retinol recovery overage is 90%. DBS retinol concentration decreased during 7 days after sampling, it is necessary to wait this delay before to determine vitamin A concentrations. In Congolese children DBS retinol measurement showed a severe vitamin A deficiency in 8% of them. This percentage is closely correlated with clinical parameters.

[Colorimetric micromethod for serum iodine determination]. / Dosage de l'iode sérique par microméthode colorimétrique: mise au point analytique et application clinique.

The measurement of iodine is a widely accepted method to explore iodine disorders. The most precise estimation is the determination of the urinary iodine in 24-hour collections. Urine collection is notoriously difficult to obtain, specially in children. In these conditions, serum measurement could be a method to overcome these limitations. We describe a colorimetric method adapted on a microtiter plate, with optimized serum mineralization conditions. The method is linear to 2400 nmol/L with a detection limit of 75 nmol/L. Precision is below 10%. The method was validated against one automatic technique. We conclude that this relatively simple method could be an additional tool to explore dysthyroidism.

Simultaneous determination of total plasma glutathione, homocysteine, cysteinylglycine, and methionine by high-performance liquid chromatography with electrochemical detection.

We here describe an ion-exchange high-performance liquid chromatography technique with electrochemical detection for rapid quantification of glutathione, homocysteine, cysteinylglycine, and methionine. The analytical validation of the technique showed within-assay and between-assay coefficients of variation between 3.1 and 4.3%, and 3.7 and 8.6%, respectively. Percentages of recovery for overload and dilution tests were between 87 and 120%. Detection limits were 1 micromol/L for methionine and 0.5 micromol/L for other compounds. There was no interference with any physiological and pharmacological substances possessing a thiol function. Aminothiol concentrations determined in 100 control subjects (50 women and 50 men) showed no age- or sex-rated differences for except for homocysteine which was increased (+ 28%) in oldest subjects of both sexes. In 60 patients at risk (30 with chronic renal failure, 30 with diabetes), homocysteine concentration was significantly increased. No variation in other aminothiols was observed in diabetic subjects. Methionine was decreased and cysteinylglycine was increased in patients with chronic renal failure. The present technique-rapid, easy to use, and reliable-appears suitable for routine application in the exploration of aminothiol metabolic pathways including mechanisms of hyperhomocysteinemia.

Micellar electrokinetic capillary chromatography quantification of cytosine arabinoside and its metabolite, uracil arabinoside, in human serum.

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6-O-methylguanine as an internal standard. The assay was linear from 6.25 to 200 microg/ml with a quantification limit between 3 and 6 microg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m2 body surface).

Determination of 4-hydroxyifosfamide concomitantly with ifosfamide and its dechloroethylated metabolites using gas chromatography and a nitrogen phosphorus-selective detector.

A sensitive gas chromatographic (GC)/nitrogen phosphorus detection (NPD) system was developed for the determination of the antitumor drug ifosfamide (Ifos) and its 2-dechloroethylifosfamide (2-Difos), 3-dechloroethylifosfamide (3-Difos) and 4-hydroxyifosfamide (4-OHIfos) metabolites in human blood. 4-OHIfos was analyzed after coupling with a trapping agent and was used as an indicator of isophosphoramide mustard (IPM). Ifos and its metabolites 2-DIfos, 3-DIfos, 4-OHIfos and the internal standard (trofosfamide) were extracted into chloroform and then resolved by gas chromatography using a Hewlett Packard HP5 capillary column cross-linked with 5% phenyl methyl silicone (30 m; 530 microm I.D.; 2.65 microm film thickness). Precision and accuracy of the assay were determined over a three-day period and a concentration range of 3.25-50 microg/ml for Ifos, 0.8-14 microg/ml for 2D-Ifos, 0.6-10 microg/ml for 3D-Ifos and 0.08-1.40 microg/ml for 4-OHIfos. The limit of quantitation was set at 3.25, 0.80, 0.62 and 0.08 microg/ml, respectively, for Ifos, 2-DIfos, 3-DIfos and 4-OHIfos. The intra- and inter-day coefficients of variation and accuracies were less than 20%, except for a low concentration 4-OHIfos. This assay was then used to provide pharmacokinetic data on antitumor and toxicologic effects following intravenous infusion of Ifos.

Quantification of residual dimethyl sulfoxide in supernatants of haematopoietic stem cells by capillary zone electrophoresis.

Dimethyl sulfoxide (DMSO) is a chemical compound that is used to preserve haematopoietic stem cells during freezing at -180 degrees C. As DMSO is largely removed by washing before reinjection of cells into a patient, accidents (notably cardiovascular) are infrequent. The lack of a method for evaluating the residual quantities of this product led us to develop a technique for assaying DMSO by capillary zone electrophoresis without extraction. This simple, rapid and precise technique was applied to the supernatant of cell pellets of thirteen patients before and after washing.

[High dose ifosfamide at 15 g/m2/cycle: a feasibility study in 10 patients]. / L'ifosfamide à hautes doses 15 g/m2/cure. Etude de faisabilité chez 10 patients.

Ifosfamide is one of the most efficient antimitotic in soft tissue sarcoma. To try to find a possible dose-effect, 10 patients with advanced pretraited relapsed soft tissue sarcoma received 15 g/m2/cycle ifosfamide in continuous infusion during 5 days. A pharmacokinetic study was done for 2 patients. All patients received growth factors, ondansetron and 8 clonazepam. Renal toxicity was evaluated after the first and the second cycle. Twenty two cycles were delivered to patients who have been already treated with ifosfamid (10 patients with 15 g/m2 to 54 g/m2, median 27 g/m2) or cis platinum (2 patients). No major renal or neurologic toxicity was observed; only subclinical modifications of urinary enzymes excretion were found. Two patients had visual hallucinations at the end of a cycle and just in the 2 following days; another presented a neuropathy of inferior limbs. Hematological toxicity was very limited. Pharmacokinetic study did not show induction mechanism at this dosage and with this type of administration. So ifosfamide 3 g/m2 during 5 days is feasible. The few level of complications observed is perhaps linked to the daily dose of 3 g/m2 instead of 4 g/m2 or more used in the other studies.

All-trans retinoic acid pharmacokinetics and bioavailability in acute promyelocytic leukemia: intracellular concentrations and biologic response relationship.

PURPOSE: This study investigated the in vitro pharmacologic behavior and disposition kinetics of all-trans retinoic acid (ATRA) in acute myeloid leukemic (AML) cells, their sensitivity to its differentiating effect, and the in vivo response of acute promyelocytic leukemia (APL) patients after therapy. PATIENTS AND METHODS: Fresh leukemic cells from 14 AML patients (nine APL and five non-APL), were incubated in suspension culture in the absence or presence of 10(-6) mol/L ATRA. Intracellular ATRA concentration and ATRA metabolism was determined by high-performance liquid chromatography (HPLC). RESULTS: Immediate uptake is observed with maximal intracellular levels (Cmax) achieved after 24 hours of incubation. At this time, ATRA levels were variable, ranging from 20 to 230 pmol/10(6) cells (median, 100 pmol/10(6) cells). Comparison of ATRA intracellular levels with the in vitro response of patients' cell samples as measured by the percentage of nitro blue tetrazolium (NBT)-positive cells after a 3-day incubation period allowed us to discriminate a group of APL patients (n = 6) with high Cmax (group A; median, 200 pmol/10(6) cells) and maximal differentiation at day 3 (median, 80%), and a group of patients (n = 8, three APL and five non-APL) with low Cmax (group B; median, 35 pmol/10(6) cells) and poor in vitro response (median, 40%; APL cases only). Interestingly, all APL patients, except one included in group A (rapid in vitro ATRA uptakers), achieved a complete remission. CONCLUSION: These findings suggest that intracellular ATRA concentrations are determinant for ATRA response and should be taken into account when monitoring the efficacy of ATRA differentiation therapeutic trials in malignant disorders.

Characterization of nuclear retinoic acid binding activity in sensitive leukemic cell lines: cell specific uptake of ATRA and RAR alpha protein modulation.

The diverse effects of all-trans retinoic acid (ATRA) on growth, differentiation and homeostasis of vertebrate organisms are mediated by three distinct isoforms of retinoic acid receptors (RARs). Although it is not known to what extent each RAR contributes to the different effects of ATRA, several studies have demonstrated that ATRA induced granulocytic differentiation in human myeloid leukemic cell lines is mediated by RAR alpha. In this study, we investigated ATRA binding affinity of the endogenous nuclear receptors of HL-60 and NB4 leukemic cells. Scatchard plot analysis yielded an apparent dissociation constant of 5 +/- 0.3 nM and 1400 +/- 80 receptor sites per cell in HL-60 cells, whereas the NB4 promyelocytic leukemic cell line showed a lower affinity (8.5 +/- 0.5 nM and 900 +/- 30 receptor sites per cell). Modulation of RAR alpha protein (5 fold excess) was found in NB4 cells after 24 hours ATRA exposure, whereas HL-60 cells required a 72-hour culture period to weakly increase the RAR alpha protein level. These data were closely related to the ATRA intracellular concentration and kinetics of terminal differentiation of the cells.

Simultaneous determination of all-trans and 13-cis retinoic acids and their 4-oxo metabolites by adsorption liquid chromatography after solid-phase extraction.

All-trans retinoic acid (all-trans RA), the active metabolite of vitamin A, has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL), the AML3 subtype of the FAB cytological classification. Complete remission is obtained by inducing terminal granulocytic differentiation of the leukemic cells. To study all-trans RA pharmacokinetics in patients with APL, a rapid, precise and selective high-performance liquid chromatographic (HPLC) assay was developed. This method is easy and shows good repeatability (C.V. = 8.41-12.44%), reproducibility (C.V. = 9.19-14.73%), accuracy (C.V. = 3.5-11%) and sensitivity with a detection limit of 5 pmol/ml. The analysis is performed using normal-phase HPLC in an isocratic mode with UV detection after solid-phase extraction on octadecyl (C18) columns. The mobile phase is hexane-dichloromethane-dioxane (78:18:4, v/v) containing 1% acetic acid.

Differential uptake of all-trans retinoic acid by acute promyelocytic leukemic cells: evidence for its role in retinoic acid efficacy.

All-trans retinoic acid (ATRA) has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL or AML3). Complete remission is obtained by inducing granulocytic differentiation of the leukemic cells. To date, the exact mechanism through which ATRA exerts its differentiating effect is not known. The present investigation was initiated to characterize ATRA intracellular concentrations achieved in human myeloid leukemic cells in relation to their different sensitivity to ATRA differentiating effect. During the first 24 h of incubation, a significant decrease of ATRA in the culture medium and a marked increase in the intracellular concentrations were observed. Maximal uptake by the leukemic cells was reached within minutes, with levels between 20 and 260 pmol/10(6) cells (median = 100). Interestingly, a correlation between ATRA-induced differentiation and the intracellular ATRA concentration achieved was observed. In fact, patients with intracellular levels below 60 pmol/10(6) cells defined slow uptakers, never exceeded 40% differentiated cells at day 3. On the other hand, cells with 2-4-fold higher concentration (100-250 pmol/10(6) cells) achieved 100% differentiated cells at day 3. This report suggests that intracellular ATRA concentration is a key pharmacological parameter that should be taken into account to gain further insights into ATRA sensitivity in APL patients.

Retinoic acid receptors: involvement in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL), is a homogeneous subgroup of acute myelogenous leukemias characterized by phenotypic and genetic markers. APL is associated with a reciprocal chromosomal translocation t(15,17) which has been shown to disrupt the retinoic acid receptor alpha (RAR alpha) gene. As a result, a portion of the RAR alpha gene becomes fused with a chromosome 15 locus termed PML (promyelocytic myeloid leukemia) from which chimeric PML/RAR alpha fusion mRNAs are expressed. The presence of these fusion transcripts in APL patients strongly support the hypothesis that both the t(15;17), and thus PML/RAR alpha, play a crucial role in the leukemogenesis of this disease. APL cells are specifically responsive to all-trans retinoic acid (ATRA) and this characteristic has allowed the first differentiation therapy with retinoic acid. However, failure or partial responses are observed and, though this has most frequently been reported in patients at second or third relapse. The molecular basis of the absence of ATRA response in these patients has not been determined.

Pharmacokinetics of oral all-trans retinoic acid in patients with acute promyelocytic leukemia.

It has been shown that patients with acute promyelocytic leukemia (AML3 subtype) treated with all-trans retinoic acid (all-trans RA), 45 mg/m2/day, achieve complete remission through differentiation of the leukemic clone to mature myeloid cells, which die spontaneously. The pharmacokinetics of all-trans RA given by mouth were studied in 15 AML3 patients. Blood samples were drawn for 24 h following a single oral dose of 45 mg/m2 and assayed for all-trans RA and 13-cis retinoic acid (13-cis RA) plasma concentrations by specific high-performance liquid chromatography. In one patient all-trans RA and 13-cis RA levels were below the detection limits at all times. In the other patients, the time to peak concentration of all-trans RA was between 60 and 210 min (median 90 min) after ingestion, with maximum concentrations between 0.03 and 2.5 micrograms/ml (median 0.4 micrograms/ml). These concentrations were within the in vitro differentiating concentration range of all-trans RA for these patients' cells. In nine patients, enterohepatic cycling was suggested by the presence on the concentration versus time curve of a secondary peak that occurred at meal times. The apparent plasma elimination half-life was between 16.8 and 77.4 min (median 30 min). Detectable plasma levels of 13-cis RA in 12 patients indicated in vivo isomerization of all-trans RA. Despite the high inter-individual variability of all-trans RA pharmacokinetics in these patients, high blast cell counts and failure to respond to differentiation treatment tended to be associated with low all-trans RA Cmax values and high clearance estimates.

Severe acne due to chronic amineptine overdose.

We report six women with severe acne lesions associated with taking amineptine, a tricyclic antidepressant. The lesions appeared after self-administration of high doses of the drug over long periods of time. They mainly occurred on the face, back, and thorax, but were also found on the extremities and in the perineal region. In five of the six cases, severity of cutaneous lesions appeared to be correlated with degree of overdose. The sixth patient never admitted having taken amineptine. Most of the patients had been unsuccessfully treated with isotretinoin for 18 months. In all six cases, chromatography of urinary 17-ketosteroids showed abnormal peaks and retention times which were different from those usually found for known steroids. In addition, the areas under these peaks were found to be a function of the degree of intoxication and of the clinical severity of the lesions. Mass spectrometry was used to qualitatively study urinary amineptine metabolites, disclosing compounds normally found only in trace amounts, as well as certain others heretofore not described in man. In two of the three patients who stopped taking amineptine, cutaneous lesions subsequently diminished, totally disappearing in the least severe case.