Retinyl palmitate hydrolase activity in human liver.

Retinyl palmitate hydrolase (RPH) activity was studied in human liver after the optimal conditions of the assay in normal human liver homogenates were determined. The mean activity was 118 +/- 66 nmol.min-1.g-1 (mean +/- SD) protein in liver homogenates from six children and seven adults; no correlation was found between liver hydrolase activity and the enzyme endogenous substrate, ie, liver vitamin A concentrations. RPH activity was also studied in a human model of vitamin A deficiency represented by 22 children with biliary atresia: 11 patients with vitamin A deficiency (liver vitamin A concentration less than 70 nmol/g wet wt of liver) and 11 patients with normal vitamin A status after vitamin A treatment. The enzymatic assay had to be conducted after the removal of endogenous bile salts by cholestyramine because bile salts are accumulated in the liver of children with biliary atresia. No correlation was found between RPH activity and vitamin A status.

Regulation of metabolism of retinol-binding protein by vitamin A status in children with biliary atresia.

Regulation of retinol-binding protein (RBP) by vitamin A status was studied in 43 children; 25 had biliary atresia and vitamin A deficiency, 15 had biliary atresia treated by vitamin A, and 9 control children had normal liver and vitamin A status. Vitamin A and RBP were assayed and the two forms of RBP, holo-RBP and apo-RBP, were separated in both liver and plasma. No difference in liver RBP concentrations was found between the three groups; apo-RBP was the most abundant form. Plasma RBP concentrations and the ratio of retinol to RBP were lower for vitamin A-deficient than for vitamin A-treated children. Two models could be proposed: 1) a preferential secretion of holo-RBP with variations in RBP catabolism or synthesis in vitamin A-deficient liver and 2) a continuous secretion of RBP by the liver with a rapid clearance of plasma apo-RBP in vitamin A deficiency.

Relation of the relative dose response to liver concentrations of vitamin A in generally well-nourished surgical patients.

In 12 adult, generally well-nourished, surgical patients, liver vitamin A concentrations obtained by the analysis of liver biopsies were compared with the relative dose response ( RDR ). RDR is defined as the percentage increase in plasma retinol level relative to the plasma retinol level 5 h after the oral administration of a standard oral dose (450 micrograms) of retinyl acetate. Liver vitamin A values, expressed as retinol, and corresponding RDR values were 14 micrograms/g (28%), 30 micrograms/g (15%), and 58 to 434 micrograms/g (0 to 12%). Other indicators of vitamin A nutriture, which did not correlate with liver vitamin A concentrations in this group, were serum retinol level, serum retinol-binding protein concentration, and the percentage saturation of serum retinol-binding protein. The relative distribution of fatty acyl esters in liver retinyl ester did not change with total liver reserves. The relative percentage of retinol to total liver vitamin A, however, was higher (greater than 5%) when reserves were less than or equal to 30 micrograms/g. Although the RDR cutoff point of greater than 14% suggested earlier by others is in reasonable accord with our results, a somewhat higher RDR cutoff value (possibly 20%) may agree better with the suggested criterion of a liver vitamin A concentration of 20 micrograms/g, expressed as retinol, as a minimally adequate reserve.

Vitamin A relative dose response test: validation by intravenous injection in children with liver disease.

The relative dose response (RDR) test has been proposed as a tool for estimation of total body stores of vitamin A. In this study we have validated a variation of this test in children with or without liver disease. Administration of vitamin A was done by intravenous injection of 1000 micrograms of retinyl palmitate. We conclude that the RDR test by intravenous injection performed after 5 h is a reliable and sensitive indicator of vitamin A status. Values found were greater than 20% when liver vitamin A concentration is less than 20 micrograms/g liver and less than 10% when liver vitamin A concentration is greater than 20 micrograms/g liver. We also conclude that the RDR test can be applied to evaluate the efficiency of vitamin A therapy and we confirm that plasma retinol levels should not be used to screen vitamin A status.

The correlation between liver vitamin A concentrations in micro- (needle biopsy) and macrosamples of human liver specimens obtained at autopsy.

A technique is described for the analysis by gradient-elution reverse-phase high-pressure liquid chromatography of vitamin A at 325 nm in small liver samples taken by needle biopsy. This assay, in contrast to previous procedures, allows rapid separation and quantitation of approximately 0.1 microgram retinol and retinyl esters, requires only 7 mg liver sample, and separates groups of retinyl esters. Vitamin A values determined in needle biopsy samples (7 to 70 mg) and in gross samples (1 to 6.3 g) of the same 24 autopsy specimens correlated well (R = 0.96) over the range 1 to 400 micrograms vitamin A per g liver. The ratio of total vitamin A values in micro- and macrosamples was 1.09 +/- 0.46 (SD). Total vitamin A values in macrosamples of liver determined by high-pressure liquid chromatography and by a differential spectrophotometric assay also correlated well (R = 0.91). Under suitable clinical conditions, the procedure should be useful in validating indirect techniques for assessing vitamin A status.

Short- and long-term vitamin A treatment in children with cholestasis.

Newborns have limited reserve supplies of vitamin A. Infants with chronic cholestasis are in a precarious nutritional state because of their limited ability to build these stores even though the vitamin is present in their diet. In this study, we investigated liver concentrations of vitamin A in 30 children with extrahepatic biliary atresia. We demonstrate that correction of the deficiency occurs after intramuscular administration of a water-miscible solution of retinyl palmitate (100,000 IU, or 30 mg retinol equivalent). Furthermore, we evaluated the effect of vitamin A injections on liver and blood concentrations in nine children with chronic cholestasis over a 1-y period. We conclude this treatment is efficient and is well tolerated.

Impression cytology detects subclinical vitamin A deficiency.

The vitamin A status of 16 children with normal eyes and liver disease was evaluated by plasma retinol concentration and by liver vitamin A concentration or iv relative-dose-response test. Conjunctival impression cytology was performed on each child. All children with normal vitamin A status had normal conjunctival impression cytology. All children with abnormal vitamin A status had abnormal conjunctival impression cytology despite their seemingly normal clinical ocular examination. These results suggest that impression cytology can identify individuals with physiologically significant, preclinical vitamin A deficiency.

Prevalence of malnutrition and vitamin A deficiency in the Diourbel, Fatick, and Kaolack regions of Senegal: feasibility of the method of impression cytology with transfer.

The evaluation of the prevalence of ophthalmological diseases and vitamin A deficiency in Senegalese preschool-aged children enabled us to confirm the method of impression cytology with transfer (ICT) and to assess the impact of ophthalmological diseases on the cytological appearance of conjunctival cells. A simplification of the ICT method consisted of transferring cells present on a strip of paper to a glass slide by finger pressure. Harris-Schorr staining of the sample papers confirmed the results obtained by the transfer. Inflammatory trachoma influenced the results of the test. This simple method will prove very helpful for detection of vitamin A deficiency in children in underdeveloped areas.

Prevalence of malnutrition and vitamin A deficiency in the Diourbel, Fatick, and Kaolack regions of Senegal: epidemiological study.

The purpose of our study was to determine the prevalence of protein-calorie malnutrition (PCM), ocular diseases, and vitamin A deficiency in preschool children selected at random in a rural zone of the groundnut belt of Senegal. The prevalence of PCM was 37.1% (95% CI 33.8-40.2%) according to the Waterlow classification, with a majority of stunting, and prevalence of hypovitaminosis A was estimated to be 11.4% (95% CI 9.3-13.5%) by using impression cytology. Furthermore, 19.4% (95% CI 15.8-22.0%) of the children might be defined at risk of deficiency. The prevalence of Bitot's spots was equal to 0.2% (95% CI 0.03-0.9%). A problem of PCM associated with a health-endangering vitamin A deficiency existed.

Prevalence of malnutrition and vitamin A deficiency in the Diourbel, Fatick, and Kaolack regions of Senegal: a controlled study.

Two hundred and six Senegalese preschool children included in an epidemiological study were selected by their results from impression cytology with transfer (ICT) for assessment of their nutritional state by means of biological variables and for assessment of the diagnostic values of the ICT. A problem of protein-calorie malnutrition existed (transthyretin and retinol-binding protein concentrations were low) associated with vitamin A deficiency (retinol concentrations were low). The sensitivity and specificity of the ICT defined with respect to retinol (threshold fixed at 0.35 mumol/L) varied with the classification criteria of ICT and seemed to be fairly insensitive but specific.

Vitamin A concentrations in liver determined by isotope dilution assay with tetradeuterated vitamin A and by biopsy in generally healthy adult humans.

The vitamin A status in 11 generally healthy surgical patients was estimated by measuring the dilution of a 45-mg oral dose of tetradeuterated retinyl acetate (99% pure). After purification of retinol by high-performance liquid chromatography, the ratio of 2H4-retinol:1H-retinol in plasma was measured by gas chromatography-mass spectrometry. On the basis of the observed ratios of [2H4]retinol:[1H]retinol over 19-47 d, the total body reserves and liver concentrations of vitamin A were calculated. Liver biopsy samples taken at surgery were directly analyzed for vitamin A. The correlation coefficient between calculated and measured liver vitamin A concentrations for 10 of the subjects was 0.88, and the Spearman's rank correlation coefficient was 0.95 (p less than 0.002). Thus, total body reserves of vitamin A in humans can be estimated validly in the marginal and satisfactory ranges by a benign, relatively noninvasive procedure.

Serum retinol, beta-carotene, alpha-tocopherol and cholesterol in healthy French children.

Age- and sex-specific reference intervals of data derived from a healthy paediatric population are presented for retinol, beta-carotene, alpha-tocopherol, cholesterol and related proteins in serum. Age was an important covariate for the micronutrient concentration values. Retinol was highly correlated with alpha-tocopherol and cholesterol. Strong correlation was found with both beta-carotene and cholesterol. Serum levels were considered for their appropriateness as indicators of micronutrient status.

Conjunctival impression cytology with transfer as a field-applicable indicator of vitamin A status for mass screening.

The increasing importance of vitamin A deficiency in even its mild subclinical form underlines the need for a mass screening test. Clinical, biochemical and cytological methods of assessing vitamin A deficiency in a public health setting have been described and widely used. The cytological method shows promise because it enables early detection of vitamin A deficiency. However interpretation is problematic since histopathological changes are gradual with the progressive disappearance of goblet cells and appearance of enlarged epithelial cells. The reliability and validity of the impression cytology with transfer (ICT) test were assessed in order to produce a meaningful standard for this cytological method. The ICT test was performed in Senegal on 1451 children, in the course of two surveys conducted in 1989 and 1990 in rural areas. Reliability, estimated by Cohen's kappa test for evaluating intra-reader variability, and sensitivity were highest for the abnormal-normal classification (kappa = 0.91; 95% confidence interval (CI): 0.89-0.93; and sensitivity = 74%; 95% CI 66-82%). The ICT method is a cheap, noninvasive and easy test to perform in the field. This method is also reproducible and fairly sensitive according to the abnormal-normal classification. As illustrated by our proposed 50% cutoff of abnormal cytology calculated in relation to 5% of serum retinol values below 0.35 mumol/L criterion, ICT only requires a small sample for the assessment of the overall health of a community in contrast to xerophthalmia and blood vitamin A deficiency tests.

PIP: In Senegal an ophthalmologist conducted eye examinations on 192 preschool children from 2 rural villages in Niakhar district in the Diourbel region in April 1989 and on 1259 preschool and school age children from 30 rural villages from Malicounda community in the Thies coastal region in February-March 1990 to assess the reliability and validity of the impression cytology with transfer (ICT) test as a field indicator of vitamin A status. Researchers also intended to standardize the cytological results. Cohen's kappa test revealed almost perfect agreement between the observed and expected interpretations of the conjuctival imprints for the classification abnormal-normal (kappa=0.91; 95% confidence interval [CI] for ICT=0.89-0.93). Thus the ICT test was reliable. Serum retinal concentrations indicated that the ICT test was specific for the deficient-nondeficient classification (serum retinal concentrations [SRC]=96%; 95% CI 93-97%) and fairly sensitive for the abnormal-normal classification (SRC=74%; 95% CI=66-82%). These serum retinal concentration results indicated that the ICT test was valid. The researchers proposed 2 classifications--normal and abnormal--to increase reproducibility of the ICT test. Since the ICT test can detect insufficient peripheral supply of vitamin A to the eye at an early stage and has only a 50% cut off, the ICT test can determine the public health of a community with only a small sample. Further the ICT test is a reproducible diagnostic test that is inexpensive, noninvasive, and easy to perform in the field.

Serum beta-carotene and antioxidant micronutrients in children with cancer. The 'Cancer in Children and Antioxidant Micronutrients' French Study Group.

Serum antioxidant vitamins A (retinol) and E (alpha-tocopherol), beta-carotene, zinc and selenium for 418 children with newly diagnosed malignancy were compared with those of 632 cancer-free controls. Incident cancer cases and controls were 1-16 years old and recruited in 1986-1989. Age- and sex-adjusted serum concentrations of retinol, beta-carotene and alpha-tocopherol were significantly inversely associated with cancer. In similar models, the odds ratio (OR) comparing the highest with the lowest quintile was 2.06 (95% confidence interval [CI] 1.40-3.02) for retinol, 3.87 (95% CI: 2.54-5.90) for beta-carotene, 2.15 (95% CI: 1.48-3.10) for alpha-tocopherol, 1.29 (95% CI: 0.75-2.23) for selenium, and 1.94 (95% CI: 1.17-2.23) for zinc. The cancer sites that were associated with serum beta-carotene were, in general, leukaemia, lymphoma, central nervous system, bone and renal tumours. Moreover, leukaemia was associated with low mean serum levels of retinol, selenium and zinc. Subjects with lymphoma, bone and renal tumours also had lower mean retinol and alpha-tocopherol levels than controls. Brain tumour patients had low vitamin E levels. Low serum values of antioxidant vitamins were associated with childhood neoplasm occurrence. Some site-specific effect was reported. Low peripheral nutrient levels are not considered as cancer promoters but rather as an impairment of the body's defence mechanism occurring during the cancer-related metabolic and nutritional disturbances and inflammation processes.

Assessment of vitamin A deficiency in the Republic of Djibouti.

In a countrywide survey, we assessed the prevalence of vitamin A deficiency by clinical examination and impression cytology with transfer method (ICT) in a randomized sample of 2445 subjects representative of the population of the Republic of Djibouti. A plasma retinol determination was made on a part of this sample. Results lead to believe that serious vitamin A deficiency may periodically occur in Republic of Djibouti when nutritional conditions become poor. Meanwhile a large number of children, mostly in the rural area, have a marginal vitamin A status and are exposed to a high level of risk.

Assessment of vitamin A deficiency in Republic of Malawi by impression cytology method.

During a countrywide survey, we assessed the prevalence of vitamin A deficiency by impression cytology method with transfer in a randomized sample of 650 representative of the children's population of the Republic of Malawi. A vitamin A deficiency was indicated by the results of the ophthalmic examination (XN = 1,4%; X2 = 0,2%) and the ICT test (22% with deficient cytology). Results of ICT were related to age. Vitamin A deficiency seems to be a public health problem in the Republic of Malawi.

Impression cytology with transfer: an easy method for detection of vitamin A deficiency.

We described a new method to stain epithelial cells harvested by ocular impression. The cells are immediately transferred on a glass slide after the sampling; this transfer permits a very simple staining and easier reading by light microscopy. The results are compared with those obtained with the previous techniques and confirm ocular impression as a good help for vitamin A status determination.

Vitamin A deficiency in pre-school age Congolese children during malarial attacks. Part 1: Utilisation of the impression cytology with transfer in an equatorial country.

The vitamin A status of 454 pre-school age Congolese children was evaluated by the impression cytology method with transfer (ICT) and by the determination of plasma retinol. The absence of goblet cells and the presence of enlarged epithelial cells indicate a peripheral deficit of vitamin A. A level of plasma retinol lower than 10 micrograms/dl is an indicator of vitamin A deficiency. The subjects were children in good health or suffering from malaria, measles or various infectious diseases. Advantages, disadvantages, sensitivity and specificity of the ICT are discussed. We suggest its use in a mass screening program for vitamin A deficiency in developing countries.

Vitamin A deficiency in pre-school age Congolese children during malarial attacks. Part 2: Impact of parasitic disease on vitamin A status.

The vitamin A status of 454 pre-school age Congolese children was evaluated by the impression cytology method with transfer (ICT) and by the determination of plasma retinol. During malarial attacks, the vitamin A status is abnormal (deficient or marginal) in 40.8% of the children according to the ICT test and in 37.5% of the children who have plasma levels of retinol lower than 10 micrograms/dl. The mean concentration of plasma retinol in patients during malarial attacks (14.8 +/- 9.5 micrograms/dl) is significantly lower than the values found in other subjects (31.5 +/- 14.3 micrograms/dl) (p less than 0.001). A significant correlation was established between the results of the ICT test and two biochemical parameters (retinol, transthyretin). We conclude that there is a significant relationship between vitamin A deficiency and a malarial attack.

Assessment of the vitamin A status of preschool and school age Senegalese children during a cross-sectional study.

The increasing concern about vitamin A deficiency in even its mild subclinical form has created the need for a mass screening test. Various clinical, biochemical and cytological methods for assessing the vitamin A status have been widely used but all are unsatisfactory for technical, ethical or public health reasons. The two prevalence criteria defined by the World Health Organization (WHO) for a vitamin A deficiency problem of public health significance are xerophthalmia and serum retinol concentrations. Recently we proposed a prevalence criterion for impression cytology with transfer (ICT) at the level of 50% of ICT results being abnormal. We conducted a cross-sectional survey of the prevalence of vitamin A deficiency and also undernutrition during the dry season in a random sample of 1,259 children (n = 442 for the 2-6 years and n = 817 for the 7-14 years) from a rural area in Senegal. Prevalence was 0 times and 4.2 times the WHO criteria for xerophthalmia and deficient serum retinol levels, respectively in preschool children. Abnormal ICT results were more frequent in preschool than in school children (53.4% versus 21.0%). There was an association between abnormal ICT results and stunting. Vitamin A deficiency was a public health problem in preschool children as assessed by the biochemical criterion (20.9% of serum retinol values under 0.35 mumol/l) or the cytological cut-off (53.4% of abnormal ICT results) but was also found in school children (21.0% of abnormal ICT results).