Frits A.J. Muskiet

Clinical chemistry of serotonin and metabolites

Analyses of serotonin and other 5-hydroxyindoles, such as its precursor 5-hydroxytryptophan and major metabolite 5-hydroxyindoleacetic acid (5-HIAA), are indispensable for the elucidation of their (patho)physiological roles. In clinical chemistry attention is mainly focused on the diagnosis and follow-up of carcinoid tumours. For this most laboratories routinely measure urinary 5-HIAA. More recently, measurements of serotonin in platelets and urine have been advocated. Platelet serotonin may be the most sensitive indole marker for the detection of carcinoid tumours that secrete only small amounts of serotonin and/or its precursor 5-hydroxytryptophan. Although several chromatographic techniques have emerged for the analysis of tryptophan-related indoles, HPLC with either electrochemical or fluorometric detection have become the methods of choice for their quantification. HPLC-based methods combine selectivity, sensitivity and high precision, and enable the simultaneous investigation of several metabolically related indoles. This review aims to place the analysis of indoles in biological matrices in a biochemical, physiological and clinical perspective and highlights several important steps in their chromatographic analysis and quantification.

CAPILLARY GAS-CHROMATOGRAPHIC PROFILING OF TOTAL LONG-CHAIN FATTY-ACIDS AND CHOLESTEROL IN BIOLOGICAL-MATERIALS

The profiling of total long-chain fatty acids and cholesterol in a variety of biological materials, using capillary gas chromatography with flame ionization detection, is described. The within-run precision and day-to-day precision for fifteen fatty acids and cholesterol in erythrocyte samples were investigated. Quantitative data on the analysis of amniotic fluid samples collected from women in the 30th to 38th week are given together with a correlation study on their lecithin/sphingomyelin and their palmitic acid/stearic acid ratios. In addition, the method was applied to lumbar cerebrospinal fluid, plasma, isolated leukemic blood cells and neuroblastoma tissue.

Traditionally living populations in East Africa have a mean serum 25-hydroxyvitamin D concentration of 115 nmol/l.

Cutaneous synthesis of vitamin D by exposure to UVB is the principal source of vitamin D in the human body. Our current clothing habits and reduced time spent outdoors put us at risk of many insufficiency-related diseases that are associated with calcaemic and non-calcaemic functions of vitamin D. Populations with traditional lifestyles having lifelong, year-round exposure to tropical sunlight might provide us with information on optimal vitamin D status from an evolutionary perspective. We measured the sum of serum 25-hydroxyvitamin D₂ and D₃ (25(OH)D) concentrations of thirty-five pastoral Maasai (34 (SD 10) years, 43 % male) and twenty-five Hadzabe hunter-gatherers (35 (SD 12) years, 84 % male) living in Tanzania. They have skin type VI, have a moderate degree of clothing, spend the major part of the day outdoors, but avoid direct exposure to sunlight when possible. Their 25(OH)D concentrations were measured by liquid chromatography-MS/MS. The mean serum 25(OH)D concentrations of Maasai and Hadzabe were 119 (range 58-167) and 109 (range 71-171) nmol/l, respectively. These concentrations were not related to age, sex or BMI. People with traditional lifestyles, living in the cradle of mankind, have a mean circulating 25(OH)D concentration of 115 nmol/l. Whether this concentration is optimal under the conditions of the current Western lifestyle is uncertain, and should as a possible target be investigated with concomitant appreciation of other important factors in Ca homeostasis that we have changed since the agricultural revolution.

Capillary gas chromatographic profiling of urinary, plasma and erythrocyte sugars and polyols as their trimethylsilyl derivatives, preceded by a simple and rapid prepurification method.

A capillary gas chromatographic method for the profiling of trimethylsilylated mono- and disaccharides and polyols in urine, plasma and unwashed and washed erythrocytes is described. The prepurification method is based on the moderate inhibition of the derivatization experienced in the presence of physiological amounts of inorganic salts and the relative stability of the formed trimethylsilylethers towards treatment with water and dilute hydrochloric acid. Series to series quality control data for 31 sugars/polyols in a pooled urine are given. The method was used to establish age-dependent concentrations of 18 sugars/polyols in urines of 72 control persons on a free diet. Gas chromatographic profiles and quantitative data obtained from urines of pediatric patients with galactosemia treated with a diet low in lactose and galactose, type 1 hereditary tyrosinemia treated with a diet low in phenylalanine and tyrosine, and a neurological disorder with a high calorie gastric drip feeding, are presented and discussed. Examples of the profiling of sugars/polyols in the plasma, unwashed and washed erythrocytes of a healthy adult, and the plasma of a newborn with galactosemia prior to treatment, are given.

EFFECTS OF SUPPLEMENTAL DIETARY CALCIUM ON QUANTITATIVE AND QUALITATIVE FECAL FAT EXCRETION IN MAN

Oral calcium supplementation is thought to be a useful interventional agent to decrease colon cancer risk. This is supposedly due, at least in part, to the binding of bile acids and fatty acids by calcium in the colon, thus prohibiting the damaging effects of these substances to the epithelium. To determine the effects of calcium supplementation on fecal fat excretion, 24 subjects kept a fat and calcium constant diet for one week and were supplemented with either 0, 2 or 4 g elemental calcium as calcium carbonate in a double-blind fashion. At the end of the week 72-hour feces was collected, and total fat, neutral fat, fatty acids and the ratio of polyunsaturated and saturated fatty acids (P/S ratio) were measured. Calcium dose-dependently increased the percentual excretion of total fat as related to fat intake: 6.8 +/- 0.9% during 0 g, 7.4 +/- 1.0% during 2 g and 10.2 +/- 1.4% during 4 g, r = 0.44, p = 0.03. This was due to increased fatty acid excretion, excretion of neutral fat was not affected, nor was the P/S ratio. It is concluded that calcium supplementation modestly increases fecal fatty acid excretion. No adverse metabolic effects are to be expected from this in case of long-term calcium supplementation in subjects at increased risk for colon cancer.

Estimated macronutrient and fatty acid intakes from an East African Paleolithic diet

Our genome adapts slowly to changing conditions of existence. Many diseases of civilisation result from mismatches between our Paleolithic genome and the rapidly changing environment, including our diet. The objective of the present study was to reconstruct multiple Paleolithic diets to estimate the ranges of nutrient intakes upon which humanity evolved. A database of, predominantly East African, plant and animal foods (meat/fish) was used to model multiple Paleolithic diets, using two pathophysiological constraints (i.e. protein < 35 energy % (en%) and linoleic acid (LA) >1.0 en%), at known hunter-gatherer plant/animal food intake ratios (range 70/30-30/70 en%/en%). We investigated selective and non-selective savannah, savannah/aquatic and aquatic hunter-gatherer/scavenger foraging strategies. We found (range of medians in en%) intakes of moderate-to-high protein (25-29), moderate-to-high fat (30-39) and moderate carbohydrates (39-40). The fatty acid composition was SFA (11.4-12.0), MUFA (5.6-18.5) and PUFA (8.6-15.2). The latter was high in α-linolenic acid (ALA) (3.7-4.7 en%), low in LA (2.3-3.6 en%), and high in long-chain PUFA (LCP; 4.75-25.8 g/d), LCP n-3 (2.26-17.0 g/d), LCP n-6 (2.54-8.84 g/d), ALA/LA ratio (1.12-1.64 g/g) and LCP n-3/LCP n-6 ratio (0.84-1.92 g/g). Consistent with the wide range of employed variables, nutrient intakes showed wide ranges. We conclude that compared with Western diets, Paleolithic diets contained consistently higher protein and LCP, and lower LA. These are likely to contribute to the known beneficial effects of Paleolithic-like diets, e.g. through increased satiety/satiation. Disparities between Paleolithic, contemporary and recommended intakes might be important factors underlying the aetiology of common Western diseases. Data on Paleolithic diets and lifestyle, rather than the investigation of single nutrients, might be useful for the rational design of clinical trials.

Dietary Influences on Plasma and Urinary Metanephrines: Implications for Diagnosis of Catecholamine-Producing Tumors

CONTEXT Measurements of the 3-O-methylated metabolites of catecholamines [metanephrines (MNs)] in plasma or urine are recommended for diagnosis of pheochromocytoma. It is unclear whether these tests are susceptible to dietary influences. OBJECTIVE The aim of the study was to determine the short-term influence of a catecholamine-rich diet on plasma and urinary fractionated MNs. DESIGN, SETTING, AND PARTICIPANTS We conducted a crossover study in a specialist medical center involving 26 healthy adults. INTERVENTIONS Subjects consumed catecholamine-rich nuts and fruits at fixed times on one day (about 35 mumol dopamine and 1 mumol norepinephrine) and catecholamine-poor products on another day. Blood and urine samples were collected at timed intervals before, during, and after experimental and control interventions. MAIN OUTCOME MEASURES Isotope-dilution mass spectrometry-based measurements of plasma and urinary concentrations of free and deconjugated 3-methoxytyramine (3-MT), normetanephrine (NMN), and MN were made. RESULTS The catecholamine-rich diet had substantial effects (up to 3-fold increases) on plasma concentrations and urinary outputs of free and deconjugated 3-MT. Dietary catecholamines had negligible influences on free NMN in plasma and urine, but substantial effects (up to 2-fold increases) on deconjugated NMN in plasma and urine. Concentrations of free and deconjugated MN in plasma and urine remained unaffected. CONCLUSIONS Dietary restrictions should be considered to minimize false-positive results for urinary and plasma deconjugated MNs during diagnosis of pheochromocytoma. Similar considerations appear warranted for plasma and urinary free 3-MT, but not for free NMN or MN, indicating advantages of measurements of the free compared to deconjugated metabolites.

Supplementation of a low dose of DHA or DHA+AA does not prevent peripartum depressive symptoms in a small population based sample.

BACKGROUND The decrease of maternal docosahexaenoic (DHA) status during pregnancy has been associated with postpartum depression, especially in women with a low intake of DHA. Since the DHA intake in the Netherlands is low, we investigated whether supplementation of low doses of DHA or DHA plus arachidonic acid (AA) during pregnancy and lactation could prevent depressive symptoms and sleep disturbances in this period. METHODS Women were supplemented daily with placebo, DHA (220 mg) or DHA+AA (220 mg each) from week 16 of pregnancy till three months postpartum. Fatty acid analyses were performed in the available plasma samples at 16 and 36 weeks of pregnancy. Depressive symptoms were measured in weeks 16 and 36 of pregnancy and six weeks postpartum using EPDS and within one week postpartum using a blues questionnaire. RESULTS 119 women completed the study. The average frequency of fish intake was low, 0.94 times per week, and did not differ between the groups. The supplementation groups did not differ in mean EPDS scores or changes in EPDS scores, nor in incidence or severity of postpartum blues. Red blood cell DHA, AA and DHA/AA ratio did not correlate with EPDS or blues scores. Indices of sleep quality did not differ between the groups. CONCLUSION Supplementation of 220 mg/day DHA or DHA+AA (220 mg/day each) does not prevent peri-partum depressive symptoms, in a population based sample with low background DHA intake. TRIAL REGISTRATION ISRCTN Register nr. ISRCTN58176213.

Neurologic condition of healthy term infants at 18 months: Positive association with venous umbilical DHA status and negative association with umbilical trans-fatty acids

Prenatal long-chain polyunsaturated fatty acids (LCPUFAs) and trans-fatty acids may affect neurodevelopment. In healthy term children, we determined relationships between relative fatty acid contents of umbilical arteries and veins and neurodevelopment at 18 mo. The study comprised a mixed group of 317 breast-fed, formula-fed, and LCPUFA formula-fed children. Study endpoints were the Hempel neurologic examination resulting in a neurologic classification and neurologic optimality score (NOS), and the Bayley Psychomotor Developmental Index (PDI) and Mental Developmental Index (MDI). Fifteen children showed minor neurologic dysfunction (MND). The umbilical vein trans, trans-18:2n-6 content was higher in children with MND than in the normal group. The NOS was significantly reduced in infants with an umbilical vein docosahexaenoic acid (DHA) content within the lowest quartile. Umbilical vein arachidonic acid (AA) was related to NOS in univariate statistics but not in multivariate analyses. The sum of trans-fatty acids and that of C18 trans-fatty acids showed a negative association with NOS in both univariate and multivariate analyses. No associations were found between AA, DHA and total trans-fatty acids with PDI or MDI. In conclusion, neonates with a relatively low DHA status and those with high trans-fatty acid levels have a less favorable neurologic condition at 18 mo.

Evidence for a metabolic shift of arginine metabolism in sickle cell disease

Over the last few years, a pivotal role has been ascribed to reduced nitric oxide (NO) availability as a contributing factor to the vaso-occlusive process of sickle cell disease. We investigated whether arginine metabolism in sickle cell patients is different from healthy controls. Blood samples were drawn by venipuncture in the fasting state from 8 clinically asymptomatic HbSS patients and 14 race-matched HbAA controls. HbSS patients had decreased plasma arginine (p=0.001) and increased proline (p=0.015) levels as compared to controls. Ratios of arginine to ornithine (p<0.001), proline (p<0.001), glutamate (p=0.003), and citrulline (p=0.026) were lower in HbSS patients. There were significant correlations of ornithine (rs=−0.71, p=0.047), citrulline (rs=−0.79, p=0.021), arginine/ornithine (rs=0.93, p=0.001), and arginine/citrulline (rs=0.81, p=0.015) to hemoglobin and of arginine/proline (rs=−0.76, p=0.028) and citrulline (rs=0.71, p=0.048) to leukocyte counts. These data indicate that in clinically asymptomatic sickle cell patients increased arginine metabolism is shifted to the arginase pathway and that this seems to be more profound in patients with higher hemolytic rates and leukocyte counts.