Plasma fatty acid status in Moroccan children: increased lipid peroxidation and impaired polyunsaturated fatty acid metabolism in protein-calorie malnutrition

Plasma fatty acid status in Moroccan children: increased lipid peroxidation and impaired polyunsaturated fatty acid metabolism in protein-calorie malnutrition

Biomed Pharmacother 2001 ; 55 : 155-62 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0753332201000415/FLA Original ar...

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Biomed Pharmacother 2001 ; 55 : 155-62 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0753332201000415/FLA

Original article

Plasma fatty acid status in Moroccan children: increased lipid peroxidation and impaired polyunsaturated fatty acid metabolism in protein-calorie malnutrition F.Z. Squali Houssaïni1, T. Foulon2*, N. Payen2, M.R. Iraqi3, J. Arnaud4, P. Groslambert2 1 Laboratoire de Biochimie, Faculté des Sciences, Dhar Mehraz, BP 1796, Atlas, Fès, Morocco; 2Laboratoire de Biochimie A, CHU Grenoble, BP 217, 38043 Grenoble cedex, France; 3Unité de Gastro-Entérologie Pédiatrique, 34 Boulevard Mohamed V, Fès, Morocco; 4Laboratoire de Biochimie C, CHU Grenoble, BP 217, 38043 Grenoble cedex 9, France

(Received 12 October 2000; accepted 31 October 2000)

Summary – In previous studies on plasma fatty acid and antioxidant status in 29 malnourished Moroccan children (12 with mild protein-calorie malnutrition, 17 with severe protein-calorie malnutrition) compared to 15 healthy control children from the same area, we pointed out that these populations were heterogeneous in terms of their essential fatty acid and antioxidant status. The aim of the present study was to classify the children using the Waterlow classification and their essential fatty acid status. The discrepancies in lipid parameters, nutritional and inflammatory markers, blood oxidative indexes, antioxidant micronutrients or trace elements (selenium, zinc, vitamin E) related to polyunsaturated fatty acids were checked in these populations. Eight of the control subjects and nine of the severe protein-calorie malnutrition children were essential fatty acid-deficient, compared to only one of the mild protein-calorie malnutrition group. Examination of the essential fatty acid-sufficient subjects with mild protein-calorie malnutrition, compared to the essential fatty acid-sufficient control subjects, showed only a decrease in Z scores and a non-significant decrease in selenium and vitamin E. In severely malnourished children, albumin, cholesterol and low density lipoprotein (LDL) cholesterol, plasma selenium, vitamin E and zinc were low, whereas inflammatory proteins and triglycerides were high. These features worsened with essential fatty acid deficiency. In all protein-calorie malnutrition subjects, there was oxidative stress (increase in thiobarbituric-acid reactants, imbalance between plasma polyunsaturated fatty acid, vitamin E and selenium levels), even in the absence of essential fatty acid deficiency. Monounsaturated fatty acids, oleic acid/stearic acid (C18:1 n-9/C18:0) ∆9 desaturase and n-3 and n-6 elongase activity indexes increased. The C18:1/C18:0 ∆9 desaturase activity index was negatively correlated to Z scores (r = –0.44, P < 0.01 for Z score weight, r = –0.39, P < 0.01 for Z score height), albumin (r = –0.82, P < 0.01) and zinc (r = –0.51, P < 0.01) levels. In essential fatty acid-deficient, severe protein-calorie malnutrition subjects, ∆6 desaturase activity was impaired, and there was a non-significant decrease in arachidonic acid. Essential fatty acid deficiency is a type of malnutrition, and is associated with an aggravation of all parameters in severe protein-calorie malnutrition. The increase in the C18:1/C18:0 ∆9 desaturase activity and enhanced lipid peroxidation without any essential fatty acid deficiency could be early markers of protein-calorie malnutrition. © 2001 Éditions scientifiques et médicales Elsevier SAS child / fatty acids / inflammation / lipid peroxidation / protein-calorie malnutrition / selenium / vitamin E / zinc *Correspondence and reprints.

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In Morocco, infantile malnutrition is a public health problem. We previously compared the oxidative balance of Moroccan children suffering from proteincalorie malnutrition (PCM) to that of healthy Moroccan children from the same area and found a decrease in antioxidant vitamins (retinol, alpha-tocopherol and carotenoids), trace elements (serum zinc, copper and selenium), and an increase in thiobarbituric-acid reactants (TBARs) in PCM children. This oxidative imbalance was associated with an increase in the prognostic inflammatory and nutritional index (PINI); this study pointed out that the antioxidant status in several healthy control children was not optimal [1]. In these children, we also compared plasma lipids and fatty acid (FA) distribution. In severely malnourished children, we found a decrease in apolipoprotein AI, total cholesterol and low density lipoprotein (LDL) cholesterol levels, with an increase in triglyceride levels. Total plasma FA levels did not vary, but the plasma FA profile was modified, with a decrease in highly polyunsaturated fatty acids (PUFA), eicosatrienoic (C20:3 n-6), arachidonic (C20:4 n-6) and eicosapentaenoic (C20:5 n-3) acids, whereas monounsaturated fatty acids increased. The oleic acid/stearic acid (C18:1 n-9/C18:0) ∆9 desaturase and the n-3 and n-6 elongase indexes were high [2]. There was no evidence of essential fatty acid (EFA) deficiency in severe PCM children compared to control children. However, both populations were heterogeneous in terms of FA status. This finding, and the heterogeneity of the antioxidant status observed in control children persuaded us to classify control and PCM children according to their EFA status and to check the relation between lipid parameters, nutritional or inflammatory markers, blood oxidative indexes (TBARs), antioxidant micronutrients or trace elements and FA status in these groups, in order to pinpoint the main features of PCM. PUFAs, as well as a substantial amount of saturated FAs, are of dietary origin. They play an important role in maintaining the fluidity and the function of cell membranes [3] and are involved in inflammation and immunity as precursors of eicosanoids [4-6]. Selenium and zinc, which contribute to the regulation of PUFA metabolism [7], might play a role in plasma PUFA content. On the other hand, the degree of unsaturation of FAs increases their suscep-

tibility to oxidation, and PUFAs are highly oxidizable. The amount or type of plasma antioxidant micronutrients (e.g., vitamin E, β-carotene, selenium) may influence plasma FA composition by preventing lipid peroxidation [8]. Cabré et al. [9] demonstrated that, in healthy subjects, only the antioxidant effect of selenium was related to the plasma FA status and that vitamin E, the most important circulating fat-soluble antioxidant, was directly linked to PUFA levels and related to dietary lipid supply. According to these observations, we focused our study on the relations between plasma FAs and plasma vitamin E, selenium and zinc levels in the populations studied.

MATERIALS AND METHODS

Patients We studied 44 children, aged 6 to 60 months, divided into three groups, according to Waterlow’s criteria of malnutrition [10]. Group 1 consisted of 15 healthy children (control group) aged 6.5 to 54 months (mean ± 1, SD = 16.6 ± 13.4 months) recruited from the outpatient clinic of Ain El Qadous on one of the following occasions: second dose of vitamin D at the age of 6.5 months, vaccination against measles at 9 months, or a growth checkup visit. Group 2 was made up of 12 children suffering from mild PCM (70 to 80% weight for age, 85 to 90% height for age and no edema) aged 18 to 66 months (mean ± 1, SD = 27.6 ± 14.4 months), recruited from the pediatric department of Ibn El Katib Hospital in Fès. Group 3 contained 17 children suffering from severe PCM (less than 70% weight for age, less than 85% height for age and no edema) aged 6 to 60 months (mean ± 1, SD = 19.6 ± 12 months), hospitalized in the same hospital; all of them presented associated infectious diseases. Weight and height were measured on admission and related to National Center for Health Statistics Percentiles (NCHS) standards [11-13]. This work has respected the ethics norms of the Human Experimental Committee and received the agreement of the Moroccan Ministry of Health.

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Methods Anthropometric data included weight, height and Z score calculation for weight and height. After an overnight fast, 3 mL of veinous blood was drawn on sodium EDTA, and 3 mL blood was drawn in traceelement-free heparinized tubes prepared in the laboratory and immediately chilled on ice. Plasma was separated after centrifugation at 2500 g for 20 min at 4° C and kept frozen (–20° C) for less than 4 weeks. Total plasma cholesterol (TC) and triglycerides (Tg) were measured by enzymatic methods (PAPTrinder, Merck Clevenot, Nogent sur Marne, France) on a Cobas Fara II centrifugal analyzer (Roche Diagnostics, Basel, Switzerland). High density lipoprotein cholesterol (HDL-C) was measured in the supernatant obtained after precipitation of apoprotein B containing lipoproteins with sodium phosphotungstate/Mg Cl2 (Boehringer, Mannheim, Germany). LDL cholesterol (LDL-C) was calculated according to Friedewald’s formula [14]. Apolipoprotein AI was measured by immunoturbidimetry on a Cobas Fara II centrifugal analyzer (Orion reagents, Epsoo, Finland). Fatty acids Lipids were extracted by a monophasic system: Hexane/isopropanol 3/2 v/v [15, 16], after addition of heptadecanoic acid as an internal standard. Extracted lipids were saponified then methylated with boron trifluoride 14% in methanol [17]. After extraction, the methylated fatty acids were quantified by gas liquid chromatography with flame ionisation detection (Hewlett Packard 5890 A with a model HP3396A integrator) on a capillary column (Alltech, Deerfield, IL, USA: AT-WAX polar 30 m length, 0.25 mm id, film thickness 0.25 µm). Hydrogen was the carrier gas; the temperature was programmed at 2° C/min increments from 168 to 235° C. The peaks of FA were identified and quantified by comparison to a mixture of known and calibrated standards and expressed as relative and absolute values. The activity indexes of ∆6 and ∆9 desaturases (dihomoγlinolenic acid/linoleic acid:20:3 n-6 / 18:2 n-6 and oleic acid/stearic acid: 18:1 n-9 / 18:0, respectively), n-3 and n-6 elongases (docosapentaenoic acid/eicosapentaenoic acid 22:5 n-3 / 20:5 n-3 and docosatetraenoic acid/arachidonic acid 22:4 n-6 / 20:4 n-6, respectively) were estimated by the derived/

precursor ratio [18]. The {n-7 (palmitoleic acid + cis vaccenic acid 16:1 + 18:1)} / {18:2 n-6} ratio was calculated to estimate an essential fatty acid (EFA) deficiency [18]. This ratio was used to divide the populations into EFA-sufficient (EFAS) and EFAdeficient (EFAD) subjects. Thiobarbituric acid reactants were evaluated in plasma by fluorescence using a fluorometer (model L50, Perkin-Elmer Ltd, Norwalk CT, USA) with a malondialdehyde kit (Sobioda, Grenoble, France) as described previously [19]. Trace elements Electrothermal atomic absorption spectrometry was used to determine the plasma selenium concentration with a Perkin-Elmer Zeeman model 5100 device. Zinc concentration was determined by flame atomic absorption spectrophotometry with a Perkin-Elmer model 460 device. Plasma vitamin E was determined by isocratic high-performance liquid chromatography [20]. Albumin, transthyretin (TTR) and orosomucoid were assayed by laser immunonephelometry (Automatic Nephelometer Behring [Behringwerke, Marburg, Germany]), and C reactive protein (CRP) by immunoturbidimetry (Turbitimer [Behringwerke, Marburg, Germany]). The prognostic inflammatory and nutritional index (PINI) was calculated as follows: PINI = orosomucoid (g/L) * CRP (mg/L) / albumin (g/L) * TTR (mg/L). Statistical analysis Data are expressed as the mean ± 1 SD and the median (minimum/maximum). Significance of differences was estimated by the Mann-Witney nonparametric test. The limit for statistical significance was set at P < 0.05. Pearson’s correlation coefficients were determined to assess the significance of associations between parameters. RESULTS Based on an n-7/C18:2 ratio > 0.2 (the usual upper limit evaluated in our French reference children by the mean ± 2 SD, is 0.16), the children were divided into five groups (table I): EFAS control group: N = 7, aged 22 ± 18 months (median = 11); EFAD control group: N = 8 (53% of control children), aged 12 ± 4

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Table I. Classification of children according to their EFA status. Fatty acid

Control subjects EFAS N = 7 EFAD N = 8

Mild PCM subjects EFAS N =11

Severe PCM subjects EFAS N = 8 EFAD N = 9

p2

p3

0.12 ± 0.07 0.28 ± 0.19*** 0.021 0.12 (0.09–0.15) 0.21 (0.17–0.76)

0.001

NS

0.04 ± 0.06 0 (0–0.13)

0.045

0.082

NS

NS

NS

0.016

0.003

NS

n-7/C18:2

0.07 ± 0.01 0.26 ± 0.07 *** 0.10 ± 0.03 0.07 (0.05–0.08) 0.24 (0.21–0.41) 0.10 (0.06–0.15)

C20:3 n-9%

0 0

C16:0%

20.03 ± 1.94 26.95 ± 2.41 *** 19.5 ± 2.4 19.8 (16.9–22.6) 27 (22.6–30.6) 18.8 (16.4–23.3)

21.1 ± 1.1 24.5 ± 1.1*** 21.3 (19.5–22.7) 24.7 (22.9–26.5)

C16:1n-7%

1.12 ± 0.25 1.16 (0.65–1.43) 15.8 ± 2.5 14.8 (12.1–19) 1.38 ± 0.12 1.33 (1.27–1.6)

3.83 ± 0.57 *** 3.63 (3.26–4.82) 22.8 ± 1.9 ** 22.6 (19.5–25.5) 1.72 ± 0.18 ** 1.68 (1.52–2.02)

1.64 ± 0.53 1.62 (0.93–2.48) 20.7 ± 3.8 20.4 (15.1–27.1) 1.59 ± 0.19 1.65 (1.29–1.85)

1.93 ± 0.44 1.92 (1.25–2.60) 25.6 ± 4.1 25.4 (20.6–33) 1.66 ± 0.11 1.63 (1.48–1.83)

C18:2n-6%

38.2 ± 3.3 38 (22.4–42.4)

21.7 ± 2.9 *** 21.7 (16.3–25)

33.8 ± 6.0 33.3 (25.8–45)

30.2 ± 2.9 22.6 ± 5.0** 30.9 (24.5–33.8) 24.1 (6.6–28.4)

C18:3n-3%

1.36 ± 0.51 0.64 ± 0.14 ** 1.18 (0.83–2.13) 0.6 (0.5–0.9)

1.13 ± 0.48 1.06 (0.59–2.32)

1.03 ± 0.59 0.9 (0.5–2.4)

PUFA %

51.5 ± 5.0 33.7 ± 4.4 *** 48.5 ± 6.4 51.2 (33.2–58.1) 33.8 (26.6–40.8) 49.3 (39.7–58.9)

C18:1 n-9% C18:1n-7%

0.25 ± 0.15 *** 0.28 (0–0.47)

0.08 ± 0.11 0 (0–0.31)

0.23 ± 0.32* 0.18 (0–1.04)

p1

3.56 ± 1.10** 0.063 3.39 (2.40–5.80) 28.6 ± 6.0* 0.006 28.2 (22.8–41.5) 1.91 ± 0.28 0.021 1.84 (1.55–2.35)

0.001 0.016 0.004

NS

0.077

0.002

NS

0.74 ± 0.29 0.76 (0.27–1.1)

NS

NS

NS

41.8 ± 3.9 33.0 ± 6.9** 42.9 (35.7–46.2) 35 (16.8–39.9)

NS

0.006

NS

Delta 6 desaturase activity index C20:3/C18:2 0.03 ± 0.01 0.08 ± 0.02 *** 0.04 ± 0.02 0.03 (0.02–0.04) 0.08 (0.05–0.12) 0.04 (0.02–0.08)

0.03 ± 0.02 0.05 ± 0.02 0.03 (0.01–0.06) 0.04 (0.02–0.12)

NS

NS

0.021

Delta 9 desaturase activity index C16:1/C16:0 0.060 ± 0.010 0.140 ± 0.020** 0.06 (0.03–0.06) 0.14 (0.12–0.19) C18:1/C18:0 2.33 ± 0.34 3.30 ± 0.50 *** 2.17 (1.9–2.74) 3.11 (2.86–4.16)

0.091 ± 0.021 0.09 (0.06–0.12) 4.04 ± 0.87 3.88 (2.92–5.65)

0.080 ± 0.020 0.08 (0.06–0.11) 2.97 ± 0.46 3.02 (2.19–3.65)

0.145 ± 0.044** 0.01 0.002 NS 0.14 (0.09–0.23) 4.73 ± 1.23 0.016 <0.001 0.021 5.02 (3.01–6.46)

Values are the mean ± 1 SD, median (minimum–maximum). EFAS: essential fatty acid-sufficient; EFAD: essential fatty acid-deficient (n-7/ C18: 2 > 0.2, C20: 3 n-9 > 0.2%). Differences tested by the nonparametric Mann-Whitney test. EFAS / EFAD P < 0.05: *; P < 0.01: **; P < 0.001: ***. p1: EFAS controls vs EFAS mild PCM; p2: EFAS controls vs EFAS severe PCM; p3: EFAD controls vs EFAD severe PCM.

months (median = 11.5) with an increase in C20:3 n-9 (Mead acid), palmitic (C16:0) and monounsaturated fatty acids, a decrease in EFAs, linoleic (C18:2 n-6) and linolenic (C18:3 n-3) acids, and in total PUFAs, an increase in ∆6 and ∆9 desaturase activity indexes. These phenomena are characteristic of the EFA deficiency [18, 21]; EFAS children with mild PCM: N = 11 aged 28 ± 16 months (median = 24); EFAS children with severe PCM: N = 8 aged 20 ± 7 months (median = 19); EFAD children with severe PCM: N = 9 (53% of severe PCM children) aged 19 ± 16 months (median = 18), with the same characteristics of EFAD control subjects, except for the absence of increase in the ∆6 desaturase activity index. EFA status in EFAS mild was less optimal than in EFAS control subjects, with higher n-7/C18:2 ratio

(P = 0.021), monounsaturated fatty acid levels (P = 0.006 for C18:1 n-9 and 0.021 for C18:1 n-7) and ∆9 desaturase activity indexes (P = 0.01 for C16:1/C16:0 and P = 0.016 for C18:1/C18:0). Total fatty acid levels were comparable in the five groups (table II). In EFAD control subjects only, C20:3 n-6 increased and C22:6 n-3 decreased. C20:5 n-3 was lower in severe PCM subjects than in the control groups. The C22:4 n-6 and the n-6 elongase activity index, higher in EFAD than in EFAS control subjects, increased in all PCM subjects. The n-3 elongase activity index increased in all PCM children. The anthropometric characteristics and the nutritional and inflammatory status of the five groups are summarized in table III. All control group children’s

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Table II. Fatty acid levels in EFAS and EFAD children. FA

Control subjects EFAD N = 8

Mild PCM subjects EFAS N =11

EFAS N = 8

EFAD N = 9

8.72 ± 1.97 8.23 (6.1–12) 2.74 ± 0.52** 2.81 (2–3.36) 1.64 ± 0.36 ** 1.67 (1.15–2.02) 6.01 ± 1.55 5.23 (4.53–8.14) 0.27 ± 0.06 ** 0.27 (0.19–0.4) 0.60 ± 0.20 0.5 (0.4–1) 0.60 ± 0.16 0.58 (0.37–0.89) 1.47 ± 0.44 ** 1.43 (0.89–2.3)

8.41 ± 1.53 8.47 (5.4–11.5) 3.88 ± 0.63 3.82 (3.04–4.76) 1.30 ± 0.49 1.3 (0.63–2.13) 7.22 ± 1.37 7.38 (4.82–9.6) 0.30 ± 0.06 0.32 (0.2–0.38) 0.54 ± 0.26 0.48 (0.23–1) 0.69 ± 0.13 0.7 (0.52–0.9) 2.79 ± 0.64 2.72 (1.85–3.82)

8.80 ± 1.74 8.7 (6.5–12.4) 3.55 ± 0.83 3.58 (2.62–5.26) 0.97 ± 0.45 0.86 (0.44–1.64) 5.37 ± 1.18 5.52 (3.58–6.86) 0.26 ± 0.06 0.25 (0.18–0.36) 0.36 ± 0.13 0.32 (0.22–0.58) 0.61 ± 0.21 0.52 (0.4–0.94) 2.40 ± 0.90 2.2 (1.36–4.14)

10.12 ± 4.77 8.95 (6.9–22.6) 3.11 ± 1.34 2.86 (1.46–6.41) 1.02 ± 0.39 0.95 (0.6–1.6) 4.70 ± 1.91 4.7 (2.09–8.66) 0.24 ± 0.08 0.24 (0.08–0.35) 0.44 ± 0.29 0.34 (0.14–1) 0.61 ± 0.19 0.6 (0.3–0.9) 1.89 ± 0.71 2.08 (0.81–2.75)

C22:4/C20:4

n-6 elongase activity index 0.03 ± 0.00 0.05 ± 0.01 ** 0.03 (0.02–0.04) 0.05 (0.03–0.06)

0.04 ± 0.01 0.04 (0.03–0.07)

0.05 ± 0.01 0.05 (0.04–0.06)

C22:5/C20:5

n-3 elongase activity index 0.86 ± 0.36 1.04 ± 0.17 0.89 (0.48–1.29) 1 (0.88–1.29)

1.59 ± 0.90 1.42 (0.73–3.91)

1.87 ± 0.86 1.56 (0.82–3.36)

EFAS N = 7 8.23 ± 4.77 8.34 (5.95–10.1) PUFA mmol/L 4.16 ± 0.90 4.21 (2.95–5.25) C20:3n-6% 1.10 ± 0.19 1.11 (0.82–1.34) C20:4n-6% 6.61 ± 1.24 6.5 (4.87–8.9) C22:4n-6% 0.18 ± 0.05 0.17 (0.13–0.24) C20:5n-3% 0.76 ± 0.45 0.54 (0.36–1.41) C22:5n-3% 0.54 ± 0.14 0.53 (0.32–0.7) C22:6n-3% 2.93 ± 0.56 2.85 (1.31–3.94)

AGT mmol/L

Severe PCM subjects

p1

p2

p3

NS

NS

NS

NS

NS

NS

NS

NS

0.021

NS

NS

NS

0.023

0.032

NS

NS

0.024

NS

0.056

NS

NS

NS

NS

NS

0.06 ± 0.02 0.05 (0.02–0.1)

0.001

0.001

NS

1.78 ± 0.89 1.93 (0.79–3.43)

0.052

0.001

NS

Values are the mean ± 1 SD, the median (minimum–maximum). EFAS: essential fatty acid-sufficient; EFAD: essential fatty acid-deficient (n-7/C18: 2 > 0.2, C20: 3 n-9 > 0.2%). Differences tested by the nonparametric Mann-Whitney test. EFAS / EFAD P < 0.05: *; P < 0.01: **; P < 0.001: ***. p1: EFAS controls / EFAS mild PCM; p2: EFAS controls /EFAS severe PCM; p3: EFAD controls / EFAD severe PCM. Table III. Anthropometric, nutritional and inflammatory parameters. Parameter

EFAS Control group N=7

EFAD Control group N=8

EFAS Mild PCM group N = 11

p1

p2

p3

0.07 ± 0.72 –0.13 (–0.87–1.16)

Z–score height. DS

0.34 ± 0.71 0.23 (–0.45–1.11)

0.27 ± 0.52 0.11 (–0.22–1.11)

– 1.14 ± 0.99 –0.85 (–2.63–0)

– 1.85 ± 0.57 –2.2 (–2.41–0.97)

– 1.83 ± 0.57 –1.85 (–2.7–1.1)

48.8 ± 2.1 50 (45–50)

47.0 ± 4.7 49.4 (47.5–50)

46.9 ± 3.4 46.3 (41.3–52)

34.9 ± 6.9 35.5 (23.6–45.9)

26.4 ± 6.0* 25.5 (15.7–35.2)

NS

0.002 0.003

0.94 ± 0.43 0.66 (0.56–1.56)

1.34 ± 0.38 1.24 (0.84–1.78)

0.99 ± 0.44 1.13 (0.27–2.16)

1.94 ± 0.89 2.06 (0.91–3)

2.44 ± 0.65 2.34 (1.46–3.68)

NS

0.028 0.008

0.001 ± 0.001 0 (0–0.003)

0.001 ± 0.001 0.001 (0–0.002)

0.002 ± 0.002 0.010 ± 0.016 0.060 ± 0.130 0.002 (0–0.007) 0.003 (0.001–0.045) 0.018 (0.002–0.38)

NS

0.035 0.005

Orosomucoid (g/l) PINI °

– 1.99 ± 0.44 –2 (–2.64–1.22)

EFAD Severe PCM group N=9

Z–score weight. DS

Albumin (g/l)

0.20 ± 0.50 – 1.52 ± 0.68 0.34 (–0.73–0.88) –1.48 (–2.64–0.58)

EFAS Severe PCM group N=8

– 2.05 ± 0.48 0.001 0.001 0.001 –2.12 (–2.64–1.1) 0.013 0.003 0.001

Values are the mean ± 1 SD, median (minimum–maximum). PINI°= (orosomucoid g/L × CRP mg/L) / (albumin g/L × transthyretin mg/L). Mann–Whitney test was performed to assess differences between groups. EFAD vs EFAD: *: P < 0.05; p1 = EFAS controls vs EFAS mild PCM; p2 = EFAS controls vs EFAS severe PCM; p3 = EFAD controls vs EFAD severe PCM

Z-scores were higher than –1, whereas all PCM children’s Z-scores were lower than –1, decreasing with the severity of the malnutrition. Nutritional and inflammatory markers did not vary in mild PCM sub-

jects, but were altered in severe PCM children, especially in the EFAD group, with a decrease in albumin and an increase in orosomucoid and in the PINI index.

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Table IV. Lipid parameters. Parameter (g/L)

EFAS Control group N=7

EFAD Control group N=8

Total cholesterol

1.28 ± 0.3 1.2 (0.99–1.76)

1.24 ± 0.26 1.15 (0.93–1.57)

LDL cholesterol

0.84 ± 0.32 0.8 (0.39–1.3)

Triglycerides Apoprotéine AI

EFAS Mild PCM group N = 11

EFAS Severe PCM group N=8

EFAD Severe PCM group N=9

p1 p2

p3

1.23 ± 0.34 1.1 (0.7–1.88)

1.02 ± 0.31 0.96 (0.59–1.45)

0.89 ± 0.33 0.96 (0.45–1.41)

NS

0.064 0.048

0.73 ± 0.23 0.66 (0.42–1.09)

0.78 ± 0.31 0.67 (0.32–1.28)

0.52 ± 0.32 0.46 (0.11–1)

0.37 ± 0.26 0.35 (0–0.86)

NS

0.083 0.008

0.95 ± 0.31 1.12 (0.57–1.32)

1.42 ± 0.69 1.14 (0.78–2.29)

1.00 ± 0.32 0.89 (0.6–1.56)

1.43 ± 0.45 1.33 (0.86–2.29)

1.95 ± 1.77 1.3 (0.96–6.6)

NS

0.049

NS

0.76 ± 0.13 0.73 (0.65–1.01)

0.75 ± 0.2 0.79 (0.43–1)

0.81 ± 0.13 0.78 (0.6–1.05)

0.68 ± 0.06 0.7 (0.58–0.77)

0.57 ± 0.26 0.52 (0.15–0.9)

NS

NS

NS

Values are the mean ± 1SD, median (minimum–maximum. EFAS: essential fatty acid-sufficient; EFAD: essential fatty acid-deficient. MannWhitney test was performed to assess differences between groups. p1 = EFAS controls vs EFAS mild PCM; p2 = EFAS controls vs EFAS severe PCM; p3 = EFAD controls vs EFAD severe PCM.

Lipid parameters are reported in Table IV. There was no significant difference between the mild PCM and control groups. In children with severe PCM, total cholesterol and LDL cholesterol levels were lower and triglycerides higher than in control subjects. Apolipoprotein AI was not significantly different between the groups. These changes were greatest in cases of severe EFAD PCM. Antioxidants and oxidation parameters are reported in Table V. In the EFAD control group, vitamin E and selenium were lower than in the EFAS control group, but there was no change in TBARs, PUFA/

selenium and PUFA/vitamin E ratios. These last parameters increased in all PCM subjects, despite the absence of a discrepency in selenium and vitamin E levels between children with mild PCM and the EFAD control subjects. In the EFAD children with severe PCM, vitamin E and selenium were the lowest and the PUFA/vitamin E ratio the highest. Zinc levels were lower in EFAS and EFAD subjects with severe PCM than in the control groups. In these latter groups, PUFA and selenium levels were correlated (r = 0.74, P < 0.01), whereas they were not in PCM children.

Table V. Antioxidants and oxidation parameters. Parameter

EFAS Control group N=7

EFAD Control group N=8

EFAS Mild PCM group N = 11

EFAS Severe PCM group N=8

EFAD Severe PCM group N=9

p1

p2

p3

Vitamin E µmol/L

17.01 ± 4.57 15.6 (11.2–25.6)

12.14 ± 4.79 12.48 (5.22–18.5)

12.57 ± 3.93 11.7 (7.83–19.9)

10.54 ± 3.23 9.75 (7.33–16.4)

8.28 ± 4.82 6.31 (4.1–18.9)

0.064 0.011

NS

Selenium µmol/L

0.82 ± 0.20 0.82 (0.47–1.13)

0.61 ± 0.06* 0.63 (0.49–0.66)

0.61 ± 0.22 0.62 (0.23–0.96)

0.54 ± 0.24 0.58 (0.23–0.93)

0.48 ± 0.21 0.48 (0.19–0.81)

0.057 0.032

NS

TBARs µmol/L

2.39 ± 0.18 2.39 (2.12–2.6)

2.41 ± 0.26 2.5 (2.08–2.7)

2.82 ± 0.26 2.84 (2.42–3.12)

2.73 ± 0.46 2.56 (2.4–3.8)

2.77 ± 0.39 2.66 (2.3–3.3)

0.007 0.052

NS

PUFA/selenium

5210 ± 1026 5610 (3850–6280)

4404 ± 794 4420 (3330–5330)

7453 ± 4126 7650 ± 3630 8156 ± 5428 6030 (4590–18130) 7575 (3950–14960) 5480 (2920–17050)

PUFA/vitamin E

249 ± 43 248 (205–325) 11.2 ± 2.3 10.6 (8.9–15.4)

246 ± 91 222 (160–410) 9.8 ± 0.3 9.9 (9.3–10)

329 ± 76 304 (221–448) 10.6 ± 1.8 10.9 (8.05–13.5)

Zinc µmol/L

353 ± 96 332 (227–510) 8.6 ± 1.8 8.5 (5.9–10.9)

430 ± 191 400 (212–790) 8.3 ± 0.9 8.3 (6.8–9.5)

NS

NS

NS

0.023 0.021 0.039 NS 0.030 0.004

Values are the mean ± 1SD, median (minimum–maximum). Mann-Whitney test was performed to assess differences between groups. EFAD vs EFAD: *: P < 0.05. p1 = EFAS controls vs EFAS mild PCM; p2 = EFAS controls vs EFAS severe PCM; p3 = EFAD controls vs EFAD severe PCM.

Protein-calorie malnutrition in Moroccan children

The C20:4 n-6 correlated positively with albumin (r = 0.66, P < 0.01) and zinc levels (r = 0.49, P < 0.01), and negatively with orosomucoid levels (r = – 0.55, P < 0.01). The C20:5 n-3 correlated positively with selenium (r = 0.40, P < 0.02). The C18:1/ C18:0 ∆9 desaturase activity index correlated negatively with albumin (r = – 0.82, P < 0.01), Z score weight and height (respectively r = – 0.44, P < 0.01 and r = – 0.39, P < 0.02) and Zinc levels (r = – 0.51, P < 0.01). DISCUSSION Plasma EFA deficiency has been evaluated according to the ratio n-7/C18:2 n-6. It has been shown to be the best discriminant, since the increase in the monounsaturated n-7 fatty acids is strongly inversely correlated to the decrease in C18:2 n-6 (r = – 0.85 in our study) [18, 21]. Plasma fatty acid composition is known to be determined by total fatty acid intake over the short term [22]. Vitamin E is the main circulating fat soluble antioxidant. Its requirements increase with PUFAs, especially linoleate intake [9], and plasma vitamin E levels closely relate to dietary PUFA supply. Selenium has been reported to be the sole variable directly related to the unsaturation index of fatty acids and its antioxidant effect has been related to plasma PUFAs [9, 23]. Therefore, selenium and vitamin E levels must be interpreted according to PUFA levels [9, 23, 24]. EFA deficiency in our control subjects was associated with decreased levels of vitamin E and selenium, but PUFA/vitamin E and PUFA/ selenium ratios remained constant and there was no increase in oxidizability markers. On the other hand, in all children with PCM, there was an imbalance between PUFAs and antioxidant micronutrients, selenium and vitamin E deficiency being more severe than PUFA deficiency. These features, associated with elevated TBAR levels in all PCM patients, illustrate the increased risk of oxidative stress. The decrease in plasma n-6 metabolites, principally C20:4 n-6 in severe PCM, has been previously described in plasma lipids of malnourished children and has been related to an impairment in the ∆6 desaturase activity [25-28].This impaired ∆6 desaturase activity could be due either to protein deficiency [25, 29, 30] or zinc deficiency [7, 31], since zinc is a cofactor in the ∆6 desaturase [7, 26, 31, 32]. In our

161

study, C20:4 n-6 was related to albumine levels, as described by Wolff et al. [33] and with zinc levels, as described by Hamilton et al. [32]. The ∆6 desaturase activity index increased in EFAD control subjects, who had neither proteic nor zinc deficiency, but did not change in EFAD children with severe PCM, who were protein- and zinc-deficient. The decrease in C20:4 n-6 could also be due to an enhanced use, as a precursor of the pro-inflammatory eicosanoids, of the E2 series [28]; the inverse correlation between C20:4 n-6 and orosomucoid suggests its participation in inflammation. The increased n-6 long chain elongation reported in chronic malnutrition [25] was observed in children suffering from PCM, but also in EFAD control children, and seems to be linked to EFA deficiency as a compensatory mechanism. The increase in the n-3 elongase activity index was only observed in PCM children and essentially linked to the decrease in eicosapentaenoic acid (EPA), which could be due to lipid peroxidation, as suggested by the positive correlation between EPA and selenium. The increase in plasma monounsaturated fatty acids, which has been described in malnutrition [26, 28], was maximum in EFAD children with severe PCM. The elevated C16:1/C16:0 ∆9 desaturase activity index, found in EFAD control subjects and EFAD children with severe PCM, has been related to EFA deficiency [18]. On the other hand, the increase in the C18:1/C18:0 ∆9 desaturase activity index has been related to EFA deficiency, but also to malnutrition and zinc deficiency [7]. In the present study, and in agreement with this observation, the C18:1/C18:0 ∆9 desaturase activity index increased not only in EFAD subjects with PCM, but also in EFAS subjects with PCM. Our results demonstrate that PCM is not necessarily associated with EFA deficiency, but that the presence of an EFA deficiency is linked to an aggravation of nutritional and inflammatory parameters. The imbalance between plasma PUFAs and antioxidant (selenium and vitamin E) levels, with the elevated in vivo peroxidation index (TBARs), present in all PCM subjects, mild or severe, EFA-deficient or -sufficient, are early markers of PCM. Impaired C18:1/C18:0 ∆9 desaturase activity was also observed in EFAS subjects with mild PCM and increased with the degree of malnutrition.

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