CORRELATIONS BETWEEN A FLUORIMETRIC and MASS FRAGMENTOGRAPHIC METHOD FOR THE DETERMINATION OF 3-METHOXY-4- HYDROXYPHENYLACETIC ACID and TWO MASS FRAGMENTOGRAPHIC METHODS FOR THE DETERMINATION OF 3-METHOXY-4- HYDROXYPHENYLETHYLENE GLYCOL IN CEREBROSPINAL FLUID

One-hundred and twenty-two lumbar cerebrospinal fluid (CSF) samples were assayed for 3-methoxy-4-hydroxyphenylacetic acid (HVA) by both a fluorimetric and mass fragmentographic method. The correlation coefficient (cc) and residual standard deviation (Syx) of the results were calculated as 0.966 and 23.3 ng/ml, respectively. If only samples containing less than 100ng/ml of HVA were considered, somewhat different values for cc and Syx were found (0.854 and 10.0 ng/ml, respectively). The data obtained by the fluorimetric method were consistently 17% lower than those obtained by the mass fragmentographic method. Spiking experiments resulted in 96.5 ± 7.8% recovery for the fluorimetric method, whereas the use of a deuterated internal standard was found to compensate completely for losses in the mass fragmentographic method. In addition the correlation between two different mass fragmentographic methods for the simultaneous determination of HVA and 3-methoxy-4-hyd-roxyphenylethylene glycol (MOPEG) in CSF was studied. The measurements were performed in different laboratories and resulted in correlation coefficients of 0.941 and 0.940 and residual standard deviations of 7.6 and 1.0 ng/ml for HVA and MOPEG, respectively. From all data we conclude that mass fragmentographic methods for the determination of catecholamine metabolites in CSF are superior to fluorimetric methods because of their selectivity, reproducibility and accuracy.     

Identification of two Thormählen-positive compounds from melanotic urine by gas chromatography—mass spectrometry

The isolation of two Thormählen-positive compounds from the urine of a patient with malignant melanoma and the elucidation of their structure by gas chromatography—mass spectrometry is described. The compounds were isolated using a poly-N-vinylpyrrolidone column and separated by preparative thin-layer chromatography. After elution they were analyzed by gas chromatography and gas chromatography—mass spectrometry as their trimethylsilyl derivatives and after hydrolysis also as their tert.-butyldimethylsilyl derivatives. The results showed the main Thormählen-positive compound A to be the glucuronide of 5-hydroxy-6-methoxyindole, whereas the minor compound AX appeared to be the glucuronide of its isomer 6-hydroxy-5-methoxyindole.     

Assessment of essential fatty acid and omega3-fatty acid status by measurement of erythrocyte 20:3omega9 (Mead acid), 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3

BACKGROUND: Early suspicion of essential fatty acid deficiency (EFAD) or omega3-deficiency may rather focus on polyunsaturated fatty acid (PUFA) or long-chain PUFA (LCP) analyses than clinical symptoms. We determined cut-off values for biochemical EFAD, omega3-and omega3/22:6omega3 [docosahexaenoic acid (DHA)]-deficiency by measurement of erythrocyte 20:3omega9 (Mead acid), 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3, respectively. METHODS: Cut-off values, based on 97.5 percentiles, derived from an apparently healthy omnivorous group (six Dominica breast-fed newborns, 32 breast-fed and 27 formula+LCP-fed Dutch low-birth-weight infants, 31 Jerusalem infants, 33 Dutch 3.5-year-old infants, 69 omnivorous Dutch adults and seven Dominica mothers) and an apparently healthy group with low dietary LCP intake (81 formula-fed Dutch low-birth-weight infants, 12 Dutch vegans). Cut-off values were evaluated by their application in an EFAD suspected group of 108, mostly malnourished, Pakistani children, three pediatric patients with chronic fat-malabsorption (abetal-ipoproteinemia, congenital jejunal and biliary atresia) and one patient with a peroxisomal beta-oxidation disorder. RESULTS: Erythrocyte 20:3omega9, 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3 proved age-dependent up to 0.2 years. Cut-off values for ages above 0.2 years were: 0.46mol% 20:3omega9 for EFAD, 0.068mol/mol 22:5omega6/20:4omega6 for omega3-deficiency, 0.22mol/mol 22:5omega6/22:6omega3 for omega3/DHA-marginality and 0.48mol/mol 22:5omega6/22:6omega3 for omega3/DHA-deficiency. Use of RBC 20:3omega9 and 22:5omega6/20:4omega6 cut-off values identified 20.4% of the Pakistani subjects as EFAD+omega3-deficient, 12.9% as EFAD+omega3-sufficient, 38.9% as EFA-sufficient+omega3-deficient and 27.8% as EFA-sufficient+omega3-sufficient. The patient with the peroxisomal disorder was classified as EFA-sufficient, omega3-sufficient (based on RBC 22:5omega6/20:4omega6) and omega3/DHA-deficient (based on RBC 22:5omega6/22:6omega3). The three other pediatric patients were classified as EFAD, omega3-deficient and omega3/DHA-deficient. CONCLUSION: Use of the combination of the present cut-off values for EFA, omega3 and omega3/DHA status assessment, as based on 97.5 percentiles, may serve for PUFA supplement intervention until better concepts have emerged.     

The effects on plasma,red cell and platelet fatty acids of taking 12 g/day of ethyl-eicosapentaenoate for 16 months: dihomogammalinolenic,arachidonic and docosahexaenoic acids and relevance to Inuit metabolism

A patient with mantle cell lymphoma took 12g/day of ethyl-eicosapentaenoate for 16 months. Compared to reference values, eicosapentaenoic and docosapentaenoic acids were elevated in plasma, red cells and platelets but docosahexaenoic acid levels were in the normal range. Arachidonic acid levels were moderately reduced but dihomogammalinolenic acid levels remained in the normal range. In spite of a long chain n-3 fatty acid intake higher than in most Inuit populations, arachidonic acid levels remained considerably higher in this patient than in the Inuit. The implications for understanding of fatty acid metabolism in humans are discussed.     

Long-chain polyunsaturated fatty acids in maternal and infant nutrition

Homo sapiens has evolved on a diet rich in alpha-linolenic acid and long chain polyunsaturated fatty acids (LCP). We have, however, gradually changed our diet from about 10,000 years ago and accelerated this change from about 100 to 200 years ago. The many dietary changes, including lower intake of omega3-fatty acids, are related to 'typically Western' diseases. After a brief introduction in essential fatty acids (EFA), LCP and their functions, this contribution discusses our present low status of notably LCPomega3 in the context of our rapidly changing diet within an evolutionary short time frame. It then focuses on the consequences in pregnancy, lactation and neonatal nutrition, as illustrated by some recent data from our group. We discuss the concept of a 'relative' EFA/LCP deficiency in the fetus as the outcome of high transplacental glucose flux. This flux may in the fetus augment de novo synthesis of fatty acids, which not only dilutes transplacentally transported EFA/LCP, but also causes competition of de novo synthesized oleic acid with linoleic acid for delta-6 desaturation. Such conditions were encountered by us in mothers with high body mass indices, diabetes mellitus and preeclampsia. The unifying factor might be compromised glucose homeostasis. In search of the milk arachidonic acid (AA) and docosahexaenoic acid (DHA) contents of our African ancestors, we investigated women in Tanzania with high intakes of freshwater fish as only animal lipid source. These women had milk AA and DHA contents that were well above present recommendations for infant formulae. Both studies stimulate rethinking of 'optimal homeostasis'. Subtle signs of dysbalanced maternal glucose homeostasis may be important and observations from current Western societies may not provide us with an adequate basis for dietary recommendations.     

Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenoic acid on breastmilk fatty acid composition

We investigated whether supplementation with arachidonic acid (20:4 omega 6; AA), or a combination of AA and docosahexaenoic acid (22:6 omega 3; DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition. Ten women were daily supplemented with 300 mg AA, eight with 300 mg AA, 110 mg eicosapentaenoic acid (20:5 omega 3; EPA) and 400 mg DHA, for one week and eight women served as unsupplemented controls. Milk samples were collected on days 0, 1 and 7. The fatty acid composition of the milk was analyzed by capillary gas chromatography with flame ionisation detection. Supplementation with AA alone had no effect on breastmilk AA, but tended to reduce EPA and DHA levels. Administration of a combination of AA, EPA and DHA tended to increase both milk AA and long chain PUFA (LCPUFA)omega 3 content. A larger simultaneous increase of milk AA, DHA and EPA than observed in the present study can probably be accomplished by the use of a combination of a lower LCPUFA omega 6/LCPUFA omega 3 ratio and higher AA, EPA and DHA dosages.     

Fatty acid compositions of preterm and term colostrum, transitional and mature milks in a sub-Saharan population with high fish intakes

BackgroundThere are no data on the fatty acid (FA) compositions of preterm and term milks for sub-Saharan African populations with advancing lactation. However, it is generally acknowledged that our ancestors evolved in sub-Saharan East-Africa, where they inhabited the land-water ecosystems.MethodsWe compared the FA-compositions of preterm (28–36 weeks) and term (37–42) colostrum (2–5 day), transitional (6–15) and mature (16–56) milks in rural African women with stable dietary habits and lifelong high freshwater fish intakes.ResultsFrom colostrum to mature milk: the median docosahexaenoic acid (DHA) content decreased from 1.11 to 0.75; and arachidonic acid (AA) from 0.93 to 0.69 g% in preterm milk. In term milk, DHA decreased from 0.81 to 0.53 and AA from 1.08 to 0.55 g%. Medium-chain saturated-FA (MCSAFA) increased from 16.9 to 33.7, and 7.92–29.0 g%, while mono-unsaturated FA (MUFA) decreased from 32.5 to 22.6, and 40.0–26.5 g%, in preterm and term milk, respectively. Consistent with the literature, preterm colostrum contained higher DHA and MCSAFA, and lower MUFA compared to term colostrum. These differences vanished rapidly with advancing lactation. MUFA and MCSAFA were inversely related.ConclusionsThe presently found DHA in preterm colostrum and mature milks and AA in premature mature milk proved the highest reported in the literature so far, as derived from analysis with capillary GC-columns. We confirmed the much higher MCSAFA and lower MUFA contents in milk of rural African, compared to Westernized women. The milk FA composition of this traditional population might show us the FA composition on which our species evolved and consequently to which our genome has become adapted to optimally support (infant) health.     

Gestational age dependent changes of the fetal brain, liver and adipose tissue fatty acid compositions in a population with high fish intakes

IntroductionThere are no data on the intrauterine fatty acid (FA) compositions of brain, liver and adipose tissue of infants born to women with high fish intakes.Subjects and methodsWe analyzed the brain (n=18), liver (n=14) and adipose tissue (n=11) FA compositions of 20 stillborn infants with different gestational ages (range 8–38 weeks) born to Tanzanian women with low linoleic acid (LA) intakes and high intakes of docosahexaenoic (DHA) and arachidonic (AA) acids from local fish.Results and discussionWith advancing gestation, brain saturated-FA (SAFA; in g/100 g FA), polyunsaturated-FA (PUFA), DHA, 20:3ω6, 22:4ω6 and 22:5ω6 increased, while monounsaturated-FA (MUFA), 20:3ω9, 22:3ω9 and AA decreased. Decreasing brain AA might be caused by increasing AA-metabolism to 20:3ω6, 22:4ω6 and 22:5ω6. In the liver, SAFA, PUFA and LA increased, while MUFA decreased with gestation. The steep increase of (mostly de novo synthesized) SAFA in adipose tissue coincided with relative decreases of MUFA, PUFA, DHA, LA and AA with advancing gestation. Compared to Western infants, the currently studied African infants had higher DHA, lower AA, and a higher DHA/AA-ratio in brain and adipose tissue, while the LA content of adipose tissue was lower.ConclusionThe low LA and high DHA and AA intakes by the mothers of these infants might support optimal α-linolenic (ALA) vs. LA competition for Δ5D and Δ6D-activities and DHA vs. AA antagonism. Conversely, the Western diet, characterized by high LA and lower DHA and AA intakes, might disturb these evolutionary conserved mechanisms aiming at an optimal ω3/ω6-balance.     

Prevalence and misdiagnosis of chronic heart failure in nursing home residents: the role of B-type natriuretic peptides

Background/objectives. Without knowing the exact CHF prevalence, chronic heart failure (CHF) occurs frequently in elderly people both inside and outside nursing homes. For a diagnosis we have to rely on physical examination and additional tests. We therefore run the risk of missing CHF diagnoses or of diagnosing CHF when we should not. Natriuretic peptide assays have emerged as a diagnostic test but their use in nursing home residents is limited. We examined the number of misdiagnoses, the CHF prevalence and the role of natriuretic peptide. Method. Residents in one centre without aphasia, cognitive impairments or metastatic cancer were screened for CHF; the natriuretic peptide levels were measured separately. Results. Of the 150 residents, 103 (64%) were included (79±11 years). The diagnosis of CHF was established in 24 of these 103 residents with NTproBNP 1871 (IQR 539 to 4262) and BNP 194 (IQR 92 to 460) pg/ml. A striking result was that of the 24 residents found to have CHF after the screening, 15 (66%) had previously been undetected: NT-proBNP 1146 (interquartile range (IQR) 228 to 3341) and BNP 200 (IQR 107 to 433) pg/ml. Moreover, in 13 out of 22 residents (62%) who had previously been thought to have CHF, the diagnosis was rejected: NT-proBNP 388 (IQR 174 to 719) and BPN 90 (IQR 35 to 128) pg/ml). Regarding the diagnostic accuracy of NT-proBNP and BNP, the optimal cut-off level of NT-proBNP was 450 pg/ml with a sensitivity of 0.71 and specificity of 0.67, and for BNP it was 100 pg/ml with a sensitivity of 0.71 and specificity of 0.70. Conclusion. Both undetected and incorrect diagnoses of CHF were common. NT-proBNP and BNP were moderately accurate at diagnosing CHF. CHF prevalence was 23%. (Neth Heart J 2008;16: 123-8.)