Oral health - a reference to malnutrition
Has conventional food any effects on the oral conditions of DS? The present study showed that children with DS had poor oral hygiene and gingival health and much greater prevalence of periodontal diseases than controls (SI; Ps. 26–28). There were no significant differences (P ˃ 0.01) in sulcus bleeding index (SBI) scores between groups, irrespective of dentition. The Quigley-Hein index (QHI) of bacterial plaque was increased by 20.6% in DS children more than controls (Table 2S), referring to periodontal problems and microbial inflammatory diseases induced by bacterial plaque. The periodontal destructions are characterised by the formation of deep periodontal pockets, associated to increased quantities of bacterial plaque and intense gingival inflammation. Oral ulceration (SI; P. 26 and Table 2S) was observed also in DS, which was painful and caused significant difficulties in eating and as a result could contribute to slow healing (SI; Ps. 40–42, and Table 5S). At its worst, this caused significant difficulties in eating and drinking (SI; Ps. 41 and 42) which refer to the need of special diet (SI; P. 77 and Table 3S).
Saliva physiochemical characteristics
The structural characterisations of whole saliva and parotid saliva such as ash weight, SFR, CO2, glucose, total protein, TN, total phosphorous (TP), IgA secretory, SCN−, CN−, EC, TDS, salinity, turbidity, colour, viscosity, and surface tension were presented in Tables 1 and 2, respectively.
Table 1
Structural characterisation of whole saliva in Down's syndrome (Level 2) compared to controls (Level 1); mean (µ) ± standard deviation (σ); repetition (n) = 5
Level | Ash wt., mg/dL | SFR, mL/min | CO2, mg/dL | Glucose, mg/dL | Total protein, g/L | TN, mg/dL (a) | TP, mg/dL |
1 | 238 ± 32 | 0.46 ± 0.17 | 34.0 ± 12.0 | 0.92 ± 0.26 | 0.94 ± 0.28 | 72.5 ± 12.1 | 82.4 ± 16.7 |
2 | 465 ± 63 | 0.22 ± 0.10 | 15.7 ± 7.87 | 2.34 ± 1.02 | 3.67 ± 0.91 | 68.3 ± 23.9 | 155 ± 42.0 |
P-value | 0.012 | 0.006 | 0.005 | 0.018 | 0.001 | 0.013 | 0.001 |
Level 1: IgA secretory [142–159 mg/dL], SCN− [17.7–60.5 mg SCN−/L] (in serum: 0.65–2.28 mg SCN−/L), CN− [0.09–0.63 mg CN−/L], Cl− [540–696 mg Cl−/L], EC [4,600-5,400 µS/cm], TDS [3270–4865 mg/L], Salinity [0.662–1.490 g/L], Turbidity [0.9–4.4 NTU], Colour [2–4 Hazen units], Viscosity (η) [1.19–1.35 cP], and Surface tension (γ) [46.7–48.2 mN/m] Level 2: IgA secretory [84–116 mg/dL], SCN− [16.8–57.1 mg SCN−/L] (in serum: 0.63–2.15 mg SCN−/L), CN− [0.07–0.42 mg CN−/L], Cl− [623–968 mg Cl−/L], EC [9,800 − 12,425 µS/cm], TDS [5419–8102 mg/L], Salinity [1.152–2.619 g/L], Turbidity [6.4–9.5 NTU], Colour [6–8 Hazen units], η [1.01–1.12 cP], γ [32.1–36.9 mN/m], and Mutans streptococci colonies (66.2%) showed high values of CFU/mL (˃ 1,000,000 S. mutans) Lower limit of detection (LOD) values for TN, TP, SCN−, CN−, EC, TDS, Salinity, Turbidity, and Colour are: 0.001 mg/dL, 0.01 µg/mL, 0.08 mg/L, 0.01 mg/L, 0.74 µS/cm, 9.14 mg/L, 2.80 mg/L, 0.056 NTU, and 0.17 Hazen units, respectively Differences of distributions in the two groups (patient-control) are presented as critical values for Mann-Whitney U test; level of significance: 5% (P = 0.05) (a) N-non protein (Controls: 11.8 ± 3.57 mg/dL and DS: 10.5 ± 2.20 mg/dL) |
Table 2
Structural characterisation of parotid saliva in Down's syndrome (Level 2) compared to controls (Level 1); µ ± σ; n = 5
Level | Ash wt., mg/dL | SFR, mL/min | CO2, mg/dL | Glucose, mg/dL | Total protein, g/L | TN, mg/dL(b) | TP, µg/mL |
1 | 99 ± 15 | 0.09 ± 0.02 | 40.2 ± 16.4 | 0.17 ± 0.05 | 0.11 ± 0.07 | 62.4 ± 18.1 | 48.1 ± 0.13 |
2 | 235 ± 39 | 0.03 ± 0.01 | 48.0 ± 19.0 | 0.42 ± 0.16 | 0.19 ± 0.12 | 54.1 ± 16.5 | 33.8 ± 18.4 |
P-value | 0.009 | 0.001 | 0.001 | 0.002 | 0.001 | 0.003 | 0.006 |
Level 1: SCN− [14.1–51.3 mg SCN−/L], CN− [0.07–0.58 mg CN−/L], Cl− [479–682 mg Cl−/L], EC [810–1750 µS/cm], TDS [1330–1907 mg/L], Salinity [0.264–0.582 g/L], Turbidity [0.3–1.8 NTU], Colour [1–2 Hazen units], η [0.98–1.03 cP], and γ [38.5–41.1 mN/m] Level 2: SCN− [13.8–47.7 mg SCN−/L], CN− [0.06–0.31 mg CN−/L], Cl− [583–908 mg Cl−/L], EC [2100–2900 µS/cm], TDS [2201–3321 mg/L], Salinity [0.472–1.069 g/L], Turbidity [6.4–9.5 NTU], Colour [6–8 Hazen units], η [0.76–0.85 cP], and γ [25.9–30.2 mN/m] LODs for TN, TP, SCN−, CN−, EC, TDS, Salinity, Turbidity, and Colour are: 0.001 mg/dL, 0.01 µg/mL, 0.08 mg/L, 0.01 mg/L, 0.74 µS/cm, 9.14 mg/L, 2.80 mg/L, 0.056 NTU, and 0.17 Hazen units, respectively (b) N-non protein (Controls: 49.0 ± 12.6 mg/dL, DS: 43.9 ± 10.6 mg/dL) Differences of distributions in the two groups (patient-control) are presented as critical values for Mann-Whitney U test; level of significance: 5% (P = 0.05) |
Further, pH, alkaline and alkaline-earth elements of whole saliva and parotid saliva were viewed in Tables 3 and 4, respectively.
Table 3
pH, alkaline and alkaline-earth elements of whole saliva in Down's syndrome (Level 2) compared with controls (Level 1); µ ± σ; n = 5
Level | pH (c) | Na, µg/Ml | K, µg/mL | Mg, µg/mL | Ca, µg/mL | Ba, µg/L | Sr, µg/L |
1 | 6.95 ± 0.72 | 87 ± 23.4 | 1142 ± 158 | 5.17 ± 0.46 | 73.9 ± 23.6 | 3.41 ± 0.88 | 6.69 ± 2.11 |
2 | 7.72 ± 0.85 | 154 ± 47 | 810 ± 112 | 12.0 ± 2.3 (d) | 152.0 ± 34.2 (d) | 68.2 ± 9.70 | 22.6 ± 5.38 |
P-value | 0.002 | 0.008 | 0.046 | 0.005 | 0.002 | 0.007 | 0.006 |
r2 | | 0.9985 | 0.9999 | 0.9990 | 0.9990 | 0.9998 | 0.9999 |
RSD | | 3.18 | 0.82 | 2.40 | 3.60 | 5.64 | 1.47 |
LOD | 0.1 | 0.12 | 0.14 | 1.22 | 0.84 | 0.10 | 0.50 |
LOQ | 0.3 | 0.39 | 0.43 | 4.50 | 6.10 | 0.37 | 1.43 |
L | | 1.02–5.75 | 1.07-50.0 | 5.10–24.3 | 6.20–59.0 | 0.41–229 | 2.06–237 |
R | | 102.2 | 98.4 | 95.7 | 94.7 | 96.2 | 98.4 |
(c) Plague pH (controls): 5.76 ± 0.32; Plague pH (DS): 7.03 ± 0.58 (t = 2.94, P = 0.001) (d) DS Ca and Mg were decreased in hair 42% and 27%, respectively compared with controls Limit of quantitation (LOQ); Linearity (L); Recovery (R, %) Differences of distributions in the two groups (patient-control) are presented as critical values for Mann-Whitney U test; level of significance: 5% (P = 0.05) |
Table 4
pH, alkaline and alkaline-earth elements of parotid saliva in Down's syndrome (Level 2) compared with controls (Level 1); µ ± σ; n = 5
Level | pH | Na, µg/Ml | K, µg/mL | Mg, µg/mL | Ca, µg/mL | Ba, µg/L | Sr, µg/L |
1 | 6.52 ± 0.68 | 105 ± 32.2 | 973 ± 125 | 3.15 ± 0.11 | 26.3 ± 10.6 | 12.7 ± 0.98 | 5.63 ± 1.47 |
2 | 7.35 ± 0.81 | 197 ± 64.0 | 687 ± 104 | 4.01 ± 0.72 | 64.0 ± 7.90 | 26.3 ± 3.96 | 17.9 ± 5.80 |
P-value | 0.002 | 0.005 | 0.027 | 0.001 | 0.001 | 0.002 | 0.003 |
Differences of distributions in the two groups (patient-control) are presented as critical values for Mann-Whitney U test; level of significance: 5% (P = 0.05) |
Heavy metals, aluminium and silicon in whole saliva and parotid saliva were obtained in Tables 5 and 6, respectively.
Table 5
Heavy metals, aluminium and silicon in whole saliva of patients with Down's syndrome (Level 2) compared with controls (Level 1); µ ± σ; concentrations expressed as µg/L; n = 5
Level | Mo | Mn | Cr | Ti | Cu | Zn | Fe | Al | Si |
1 | 1.29 ± 0.47 | 8.41 ± 2.13 | ≤ LOQ | ≤ LOQ | 484 ± 108 | 674 ± 119 | 629 ± 160 | ≤ LOQ | 108 ± 49 |
2 | 1.20 ± 0.53 | 3.59 ± 0.53 | 36.2 ± 14.8 | ≤ LOQ | 936 ± 246 | 54.6 ± 21.4 | 550 ± 78 | 1140 ± 178 | 160 ± 65 |
P-value | 0.001 | 0.001 | 0.001 | 0.017 | 0.002 | 0.001 | 0.005 | - | 0.073 |
r2 | 0.9973 | 0.9982 | 0.9996 | 0.9973 | 0.9998 | 0.9994 | 0.9989 | 0.9965 | 0.9962 |
RSD | 7.24 | 3.32 | 2.63 | 1.17 | 2.39 | 0.98 | 1.13 | 7.51 | 9.06 |
LOD | 0.12 | 0.10 | 1.22 | 0.69 | 3.37 | 0.50 | 0.70 | 1.08 | 22.4 |
LOQ | 0.37 | 0.31 | 4.15 | 2.34 | 11.5 | 1.70 | 2.30 | 3.55 | 74.8 |
L | 0.50–60.0 | 0.65-37.0 | 5.20–16.8 | 2.55–87.4 | 13.2–110 | 3.07–42.8 | 5.00-108 | 18.4–193 | 93.7–224 |
R | 95.2 | 93.7 | 99.4 | 102.6 | 97.8 | 101.4 | 100.8 | 102.0 | 107.3 |
Differences of distributions in the two groups (patient-control) are presented as critical values for Mann-Whitney U test; level of significance: 5% (P = 0.05) Bovine Liver SRM 1577b, mussel tissue SRM 2976, poplar leaves GBW07604 and freeze-dried urine SRM 2670a were used in the assessments of RSD and R of the following elements in this order: Fe, Ti, Si, and some metals (Mo, Mn, Cr, Cu, Zn, and Al), respectively LOD and LOQ were calculated from the standard deviations (σ) of the response for 1% HNO3 using criteria of three times the standard deviation and ten times the standard deviation, respectively [5, 6] |
Table 6
Heavy metals, aluminium, and silicon in parotid saliva from patients with Down's syndrome compared to controls; µ ± σ; concentrations expressed as µg/L; n = 5
Level | Mo | Mn | Cr | Ti | Cu | Zn | Fe | Al | Si |
1 | 1.08 ± 0.32 | 6.15 ± 1.28 | ≤ LOQ | ≤ LOQ | 127 ± 44.0 | 130 ± 29 | 69 ± 13.4 | ≤ LOQ | 90 ± 21 |
2 | 0.94 ± 0.10 | 1.52 ± 0.16 | 26.2 ± 9.43 | ≤ LOQ | 560 ± 81 | 47.3 ± 8.57 | 24.7 ± 3.48 | 285 ± 40.2 | ≤ LOQ |
P-value | 0.001 | 0.001 | 0.001 | 0.002 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 |
Chemical characteristics of blood, urine, and hair
With limited studies of the extent of electrolytes in biological media other than saliva (especially in DS), we analysed the contents of Al, Si, and heavy metals in blood, urine, and hair (Table 7; supplemented data kept in Table 9S).
Table 7
Heavy metals, aluminium and silicon levels in biological samples from the study population (n = 5)
Level | Mo | Mn | Cr | Ti | Cu | Zn | Fe | Al | Si |
Blood (µg/L) (e) | Blood (µg/mL) |
1 | 0.59 ± 0.14 | 3.74 ± 1.19 | ≤ LOQ | ≤ LOQ | 0.39 ± 0.18 | 0.88 ± 0.13 | 0.42 ± 0.19 | ≤ LOQ | ≤ LOQ |
2 | 0.56 ± 0.17 | 0.46 ± 0.71 | 50.1 ± 13.1 | ≤ LOQ | 1.70 ± 0.53 | 0.25 ± 0.12 | 0.20 ± 0.03 | 1.14 ± 0.47 | 0.54 ± 0.12 |
95th | 0.56–0.57 | 0.45–3.11 | 0.01–4.04 | - | 0.43–1.62 | 0.25–0.81 | 0.11–0.38 | 0.01–0.86 | 0.01–0.30 |
Urine (µg/g creatinine) (f−j) | Urine (µg/mg creatinine) |
1 | 0.26 ± 0.14 | 2.56 ± 0.75 | 3.4 ± 0.90 | ≤ LOQ | 36.4 ± 9.72 | 0.32 ± 0.19 | 0.19 ± 0.07 | 1.24 ± 0.48 | 0.39 ± 0.12 |
2 | 0.27 ± 0.19 | 8.64 ± 1.38 | 0.97 ± 0.73 | ≤ LOQ | 0.76 ± 0.68 | 0.77 ± 0.14 | 37.6 ± 12.1 | 0.17 ± 0.03 | 0.15 ± 0.02 |
95th | 0.26–0.27 | 2.72–7.95 | 0.80–3.26 | - | 0.97–15.8 | 0.41–0.72 | 0.23–28.9 | 0.19–1.21 | 0.15–0.34 |
Hair (µg/g) | Hair (µg/mg) |
1 | 1.82 ± 0.10 | 5.07 ± 3.22 | 3.07 ± 0.09 | ≤ LOQ | 2.01 ± 0.64 | 0.28 ± 0.08 | 16.2 ± 3.98 | 0.56 ± 0.04 | 0.11 ± 0.07 |
2 | 1.75 ± 0.49 | 0.85 ± 0.75 | 70.8 ± 13.6 | ≤ LOQ | 64.5 ± 9.31 | 0.09 ± 0.01 | 2.46 ± 0.79 | 0.43 ± 0.12 | 8.46 ± 5.17 |
95th | 1.75–1.80 | 0.04–7.05 | 0.10–14.3 | - | 1.29–20.8 | 0.01–0.36 | 4.35–14.9 | 0.44–0.51 | 0.11–8.34 |
Level 1: Controls (N = 74), Level 2: DS (N = 71); 5–95th percentiles: 95th (e) pH (controls): 7.32 ± 0.89; pH (DS): 6.80 ± 0.74 (t = 2.29, P = 0.003) (f) pH (controls): 6.43 ± 0.66; pH (DS): 7.16 ± 0.75 (t = 2.46, P = 0.015) (g) Urinary concentrations of heavy metals were adjusted for creatinine levels to reduce inter-individual variation of the measurements (h) Hydroxyproline (OHP)/Creat. (DS: 0.023 ± 0.003 mg/mg, Controls: 0.012 ± 0.002 mg/mg, P = 0.000); Fasting urinary OHP and creatinine (Creat.) were assayed by an OHP kit and Jaffe’s colorimetric method, correspondingly (i) Ca/Creat. (DS: 0.050 ± 0.010 mg/mg, Controls: 0.043 ± 0.004 mg/mg; P = 0.000) (j) No acute kidney injury (AKI) observed and no thrombotic thrombocytopenic purpura (TTP) registered – But still a wealth of secrets on the mechanisms involved about the gamut of electrolytes of DS urine is worthy further investigations which will enhance renal physiology. So that, we suggest studying a pathophysiological characterisation of DS patients in order to enable next generation scientists to target mechanistically the right drug and best meal for DS “a potential sponge-toxics” body, at the right time |
Major possible sources of the sharing elements in this study and their normal levels in healthy men are presented in Table 12S.
Previous trials – Non-controlled designed nutritional formulas
Thiel and Fowkes [3] were designed a nutritional formula for DS patients and this capsulated: Vitamin A (Retinol: C20H30O), vitamin B1 (thiamine: C12H17N4OS+), folate (folic acid and vitamin B9: C19H19N7O6), vitamin B12 (cobalamin: C63H88CoN14O14P), vitamin C (L-ascorbic acid: C6H8O6), iron (Fe), magnesium (Mg), manganese (Mn), zinc (Zn), carnitine (β-hydroxy-γ-N-trimethylaminobutyric acid), 3-hydroxy-4-N,N,N- trimethylaminobutyrate: C7H15NO3), carnosine (β-alanyl-L-histidine: C9H14N4O3), choline (2-hydroxy-N,N,N-trimethylethan-1-aminium: C5H14NO+), and serine (2-amino-3-hydroxypropanoic: C3H7NO3). The same team had also suggested adding excess amounts of copper (Cu), cysteine (2-amino-3-sulphhydrylpropanoic acid: C3H7NO2S), and superoxide dismutase enzyme (SOD, EC 1.15.1.1, which is already high in DS!) for no justified reasons. This team had also suggested adding excess amounts of Cu and cysteine, with no explanation or any justification of this formula.
DS good health is directly linked to the behaviour which is affected by the use of nutritional supplements, vitamins, minerals, and environmental quality. However, the vitamin-mineral regimens supposedly work regardless of the cause of the brain dysfunction. So, it is hard to believe the suggested regimens in this section can have such a general beneficial effect on brain or are related to the intelligence. Therefore, environmental factors had received our attention in this work.
Macronutrients consumption
The biochemical analyses in the preliminary stage (6-months before starting the current work) have showed a well-absorption of the biochemical contents of the pills and capsules. Nonetheless, no adverse clinical or biochemical effects were found. Conventional nutritional supplements have not enhanced any specific abilities that contribute to overall global intelligence. Thus, the design of the current experiments in this work has avoided thyroid medication.
The DS consumption of macronutrients were assessed and compared with the recommended dietary intakes in Table 8.
Table 8
Dietary macro-nutriment consumption compared with the recommended dietary intake
Nutrient | µ ± σ | Recommended Dietary Intake |
Protein (g) (k,l) | 89 ± 3.03 | - |
Lipid (g) | 75 ± 3.31 | - |
CH (g) | 305 ± 8.24 | - |
Vitamin D (µg) | 6.31 ± 0.97 | 15.0 |
Ca (mg) | 592 ± 184 | 1200 |
K (mg) | 2350 ± 506 | 4700 |
Na (mg) | 5218 ± 813 | 1500 |
P (mg) | 1103 ± 169 | 1250 |
(k) The rise in protein supply is associated with an increase in total energy intake (TEI) (l) It is optional to elevate protein intake from 1.5 g/kg/day to at least 2.0-2.5 g/kg/day [7] |
Table 8 displayed the quantitative and qualitative daily intakes of macronutrients, bone-related vitamin D, and basic minerals. Fat, protein, and CH intakes were not adequate. The protein loss could be assigned to the blisters which affect the protein synthesis for tissue repair and inflammatory processes. Fats are beneficial in this perspective, as volume-for-volume they deliver more than twice the calories of proteins and CH. A positive correlation between DS- total body fat (TBF) mass and CH tolerance was observed.
Statistical correlations between DS mineral composition and food
The statistical correlations between basic consumed foods and minerals composition in DS biological material are presented in Table 9. These can prevent DS adverse health effects.
Table 9
Spearman correlation of metals concentrations in different biological samples of DS with food consumption
Element | Sample | Bread | Milk (m) | Eggs | Chicken | Veal | Canned tuna | Canned sardines | Other canned food |
Mo | Saliva | | 0.227 | | | | | | |
| Blood | | | 0.183 | | | | | |
| Urine | | | | | | | | 0.129 |
| Hair | | | | | | | | |
Mn | Saliva | 0.119 | | 0.102 | | | | | -0.164 |
| Blood | | | | 0.135(n) | | | | |
| Urine | | | | | | 0.150(n) | 0.137(n) | |
| Hair | | | | | 0.107 | 0.162 | 0.185 | |
Cr | Saliva | 0.186 | | 0.220 | 0.151 | | | | -0.130 |
| Blood | | | | | | | | |
| Urine | | | | | | | | 0.118 |
| Hair | | | | | 0.129 | | | |
Ti | Saliva | | | | | | | | |
| Blood | | | | | | | | |
| Urine | | | | | | | | |
| Hair | | | | | | | | |
Cu | Saliva | 0.121 | | | | | 0.140 | 0.146 | 0.135 |
| Blood | | | | | | | | |
| Urine | | | | | | | | |
| Hair | | | | | | 0.131 | | |
Zn | Saliva | 0.177 | 0.162 | | | | | | |
| Blood | | | | 0.276(n) | | | | |
| Urine | | | | | -0.171 | | 0.120(n) | |
| Hair | | | | | | 0.248 | | |
Fe | Saliva | 0.165 | | 0.189 | | | | | |
| Blood | 0.262 | | 0.414 | | | 0.207 | | |
| Urine | | | | | | | | -0.253 |
| Hair | | | | 0.140 | | | | |
Al | Saliva | | | | | | 0.294 | | -0.167 |
| Blood | | | | | | | | 0.121 |
| Urine | | | | | | | | |
| Hair | | | | | | | | |
Si | Saliva | 0.144 | | | | | | | |
| Blood | | | | | | | | |
| Urine | | | | | | | | |
| Hair | | | | | | | | |
(m) Interestingly, patients were taken milk-based Ca for increasing the bioavailability of Ca due to the presence of other nutrients such as proteins fragments as seen in Table 8 and non-protein nitrogen which can increase bone modelling (n) P < 0.01 Blank cells indicate lack of statistically significant correlation. P < 0.05 if the cell has not assigned with (n) |
Nutritional status may have an effect on the biological content of minerals. The associations of the elements in blood, urine, and hair with basic meals are presented in the following series (Table 9): Cr = Cu > Mn > Al = Fe = Zn > Mo = Si > Ti, Mn = Zn > Cr = Fe = Mo > Al = Cu = Si = Ti, Mn > Cr = Cu = Fe = Zn > Al = Mo = Si = Ti, respectively. Toxicokinetic differences among the studied biological samples may account for the lack of correlations found for the selected metals in these matrixes. Blood and urine levels have reflected recent exposures especially of Cr, Cu, Al, and Si, in addition to, Mo, Mn, Zn, and Fe, correspondingly, contrary to hair content particularly of Cr, Cu, and Si which may refer to past exposures.
Table 9 has also presented different associations between saliva and its metallic content and both bread and eggs. These associations were expressed in the following series: Cr = Cu > Mn > Al = Fe = Zn > Mo = Si > Ti. Hence, saliva can be defined as a primer bioindicator for malnutrition.
Challenges in in vitro findings
Turbidity of saliva – In DS saliva, streptococcus pneumonia and haemophilus influenza had been observed in both whole saliva and parotid saliva and their media were more turbid than normal subject's saliva. Turbidity of saliva was significantly correlated to C-reactive protein (CRP) (Table 9S; r = 0.95, P < 0.001). As a suggestion, trans fats should be avoided (SI; P. 77).
Salinity of saliva and sweat – DS patients achieved higher salinity in saliva than controls (Tables 1 and 2), which is caused due to the recurrent chronic idiopathic of the salivary glands. It is also dependent upon functional derangement of the digestive apparatus. Higher DS saline saliva is associated with the deficient (or depraved appetite), a thick yellow or brownish fur, nausea, pain and heaviness in the right side (clearly, referring to liver problem), thirst, constipation, headache, confused vision, and singing in the ears. These new observations guided us to estimate for the first time the salt content in DS sweat and comparing these findings with the corresponding values of the controls. Using pilocarpine (C11H16N2O2) as sweat stimulant, the osmolality of DS was larger (214 mM/kg) than these for controls (85 mM/kg). This means that DS body fluids are roughly more saline than normal individuals, which could be due to metabolic imbalance.
Viscosity of saliva – This may influence the development of caries, that was significantly lower in DS children saliva than in controls group (Tables 1 and 2).
Saliva CO 2 – The whole saliva concentrations of CO2 were significantly (P < 0.05) lower in DS than controls, however, the situation was insignificantly reversed in parotid saliva attributed to the large range. Moreover, the whole and parotid saliva mean concentrations of CO2 were not varied enormously. Expectedly, CO2 concentrations were directly correlated with salivary pH levels. Carbonic acid (H2CO3) formed of CO2 in breath in salivary water is a key mediator in mineralisation. Initially, it would dissolve food and enamel mineral but also break down and readily release the same. Moreover, the CO2 and water may enter the salivary glands either from the blood or may be formed in the glands by aerobic respiration [8]. Subsequently, the H+ ions are conveyed to the blood while Na+ ions from the blood are transferred to the salivary glands and secreted with the HCO3− ions. This series of events may be the mechanism accounting for the rise in pH, Na+, and HCO3− levels (HCO3− concentrations were not presented in the current paper) with decreases in secretion rate. Thus, an increase in carbonic anhydrase (CA, EC 4.2.1.1) activity could be the factor responsible for the electrolyte increase.
SFR and glucose – SFR was found directly proportional to CO2 levels in this study. DS subjects secreted substantially lower overall salivary constituents into the oral cavity due to the slow SFR. However, the low level of SFR in parotid saliva may primarily refer to detective secretion from the submandibular, lingual or mucus glands. Besides, the low SFR in DS whole saliva suggested a reduction in clearance of sugar (DS glucose was 2.5 times that of controls; Tables 1 and 2) may attributed to the PhA (1.24 ± 0.22 h/day) and increasing the risk of oral disease (SI; Ps. 26–29, 67 and Table 2S). Remarkably, we also found diabetes among the relatives of DS individuals, which refers to a genetic linkage that could be existed between the tendency for nondisjunction during meiosis (induced by Hg, lead (Pb), and tin (Sn); data not shown in this paper) and the tendency to develop diabetes.
Saliva total proteins – Salivary proteins have many functions, among them, the bacterial aggregation, oxidation of hydrogen peroxide (H2O2), antiviral, antimicrobial, and antifungal activity. A total protein was significantly increased in DS whole saliva and parotid saliva than in controls. Total protein was found to correlate positively and strongly highly significant with plaque index (PI) (r = 0.98, P < 0.001) and gingival index (GI) (r = 0.96, P < 0.001). These correlations proved that total proteins had collaborated so far in the inhibition of mineral precipitation and remineralisation.
TP of saliva – To the best of our knowledge, the factors which regulate the hydroxyapatite (HAP: Ca5(PO4)3(OH)) balance are free calcium and phosphate ions. Phosphorous and calcium are directly related to caries incidence, the maturation or remineralisation of enamel, and calculus formation. The mean phosphorous concentration in saliva of the study group was more than that of the control group (1.88 times for the whole saliva) and this difference between the study and controls group was found to be highly significant (P = 0.001). This limit was increased dramatically with increasing age (t = 2.015, df = 41, P < 0.01). Albeit, TP-DS parotid saliva was significantly decreased 1.42 times that of controls (Table 2).
IgA of saliva – Secretory IgA (sIgA) in saliva is a local defence factor against caries. IgA antibodies may neutralise extracellular enzymes and reduce the initial adherence of bacteria by inhibiting sucrose (C12H22O11)-independent or sucrose-dependent streptococcal accumulation on tooth surfaces. A negative correlation between sIgA level and caries prevalence has been detected in this work. Total salivary IgA was lower in DS than in controls, but the difference was not statistically significant. Therefore, we suggest that detection of salivary sIgA levels may serve as a simple predictor of the susceptibility or resistance of DS individuals to caries formation.
TN of saliva – Ammonia (NH3) production from the metabolism of urea (CH4N2O) (Controls: 44 ± 8 mg/dL, t = 12.315, df = 65; DS: 57 ± 11 mg/dL, t = 9.218, df = 65; P < 0.001) by urease (EC 3.5.1.5) enzymes of oral bacteria has moderated plaque acidification which generally could inhibit dental caries. However, it is noticeable here that the decrease in salivary TN was correlated to the low DS dental caries, the event that worth more research.
Alkaline and alkaline-earth of saliva – Sodium and potassium have seen to play a role in the regulation of SFR. The increasing of alkaline and alkaline-earth ions concentrations except K+ ions were perhaps due to maturation or remineralisation of enamel and calculus formation (Table 2S). The alkali medium of DS saliva was enforced by lower secretion of whole saliva (73.9%) and insignificantly raising of Na+, Ca2+, Mg2+, Ba2+, and Sr2+ by 48.4% (53.3% in parotid saliva), 51.4% (58.9% in parotid saliva), 56.9% (21.4% in parotid saliva), 95.0% (51.7% in parotid saliva), 70.4% (68.5% in parotid saliva), respectively (Tables 3 and 4). The differences between the study and control groups were found to be significant (P < 0.01). These cations are expected to increase with the halogen ions in saliva (Tables 1 and 2) which refers again to saline nature of DS saliva. We think Na+ ions are reciprocated through the low primary acinar secretion apparently by both active and passive processes. The effect produced by duct cells has led to Na+ removal. More to the point, Na+ ions have been increased owing to the lack of active transport mechanism at the end of the excretory ducts. A positive correlation was found between Na+ of DS saliva and salivary buffering capacity (SBC). Contrary to these findings, K+ was decreased in DS saliva (whole and parotid), suggesting that there is an alteration in the metabolism of the duct and/or acinar cells of salivary glands of DS children. However, statistically significant (P < 0.01) and negative correlations were found between these alkali and earth-alkali concentrations and DMFT indices (i.e., -4.556 for DMFT and [Ca2+], -3.211 for DMFT and [Mg2+]). Remarkably, K+ showed a negative correlation with dental caries which was not statistically significant (P > 0.01), whereas Na+ showed a positive correlation with dental caries.
Saliva pH – In whole saliva, CO2 and SFR exhibited negative correlations with pH, contrary to Mg and TN. In parotid saliva, TN was negatively correlated with pH, opposing to CO2 and SFR which were positively correlated with pH.
The relatively higher pH of DS saliva caused by the following factors:
-Parotid saliva contained higher levels of non-specific esterase (Fig. 2S) and sodium bicarbonate (NaHCO3). These caused a change in the amount of CA which is a responsible for the distributions of the cellular and secretory elements of these glands. Notably, CA increases the production of carbonic acid (H2CO3) from 200 hr− 1 to 600,000 sec− 1 [8].
-Glandular CA contribution as a catalysing agent in the reaction of CO2 and H2O gives H2CO3. This acid spontaneously dissociates into H+ and HCO3−. Most of the H+ ions attach to Hb and other proteins (Table 9S), minimising the change in blood pH. On the other hand, the CO2 and H2O possibly diffuse into the plasma which can enter the salivary glands either from the blood or be formed in the glands by aerobic respiration [8].
-Metabolic alterations due to instabilities of salivary enzymes in patients with DS.
Subsequently, H+ ions likely convey to blood (see the lower level of pH of blood of DS patients in Table 7), while, Ca2+ and Mg2+ transfer from blood to salivary glands (Tables 3 and 4) and secret with HCO3− ions. This series of events probably forms the mechanism accounting for the rise in pH, Ca2+ and Mg2+ levels in saliva with the decrease in secretion rate.
Silicon in saliva – Silica plays the role of the substrate for the nucleation but does not inhibit the conversion of the precursors to HAP. More Si (probably Si4+) ions were detected in the whole saliva but little amount found in the parotid saliva of the controls, possibly as remaining traces result from the mouth rinse with sodium fluorosilicate (Na2SiF6) (SI; P. 32).
Aluminium in biosamples – Similar to individuals with young senile dementia of the Alzheimer's type, DS appeared to have increased absorption of Al. Absorbed Al was excreted in the biological samples (Tables 5–7) without being absorbed systemically. But the question is Al a causal genetic or environmental factor in DS? The answer is that we did not detect any specific environmental factors and no significant correlations among mother's age and their children's biological Al were observed. Therefore, we gave more space to study the Al genetic effect. To elucidate this factor, we analysed saliva in different relatives to DS individuals. Among first-degree relatives (brothers and sisters), second-degree relatives (uncles and aunts), and third-degree relatives (cousins), Al concentrations in saliva were 54.8 ± 2.33 µg/L, 28.2 ± 1.67 µg/L, and 10.8 ± 1.09 µg/L, respectively. However, no significant differences in the incidence rate have been observed. Here, it is noteworthy to suggest doing more researches on possible effects on microtubule defect, brain enzymatic systems, neurotoxicity, reproductive, developmental effects, and neurobehavioral immunological following inhalation of Al.
Heavy metals (Ti, Cr, Mn, Fe, Zn, Cu, and Mo) – Generally, studies which quantified transition metals in biofluids (SM-A), gave only limited information on the metal's subsequent biological effects, especially for the case of saliva which is continuously produced, washed, and swallowed. So that, after a deep literature survey, it can be clearly seen that there is an inadequate availability of data concerning the association of DS biological heavy metals to patients' health.
In the current study, heavy metals are presented in trace amounts in biological matrixes.
Titanium analysis in biological materials has not referred to any implications.
Chromium released to the DS' saliva, blood and hair was above the average dietary intake (Table 12S) which refers to a possible toxicity with this element. Taking into consideration that it cannot be excluded that even nontoxic concentrations of Cr could be sufficient to induce biological effects in cells (as oral mucosa). Exclusively, Cr was not related to pH but probably may cause a bitter taste in the mouth which was confirmed by DS patients. From our experience, Cr has impacted sugar metabolism through its role in the uptake of insulin (Table 9S) which also causes losses of Cr in urine (Table 7). Cr also possibly aided in lowering the low-density lipoprotein cholesterol (C27H45OH) (LDL-C) and raising low-density lipoprotein cholesterol (HDL-C) (Table 9S).
Manganese is an essential element in many metabolic pathways in very limited amounts (Tables 5–7). Our results found Mn exposure has increased Fe concentration in DS biological fluids more than controls. Thus, we can say: “Mn exposure or in dose may inhibit DS-Fe absorption”.
Iron is reduced in DS saliva which may be expressed with the ineffective removal of plaque (see PI and QHI in Table 2S) and debris (see DI-S in Table 2S) from DS teeth. Therefore, it can be deduced that the decreased constricting power of pharyngeal musculature and dysphagia (sideropenic dysphagia) in DS is attributed to the reduction in the amount of Fe in their biological materials as saliva, blood, and hair. As a result, an introducing supplemental Fe for the adequate production of red blood cells and for increasing muscles masses of DS patients is highly suggested.
Zinc is an essential element for many body functions, including enzyme activity, gene expression, intestinal epithelial regeneration, male reproductive system and a variety of immune mechanisms. Zn2+ ions (like many ions as Mg2+) are moved to the salivary fluid by passive transport and play a part of the cytosolic copper-zinc SOD enzyme. Zn2+ has negatively correlated to DMFT and dental indices which were statistically insignificant (P > 0.01), so dietary Zn2+ can reduce the susceptibility to dental caries in some critical conditions. On the other hand, the lower limits of Zn in DS patients can be handled by taking Zn-containing supplements, as zinc gluconate (C12H22O14Zn) that holds very little amounts of cadmium (Cd). However, many Zn products contain Cd, this is because Zn and Cd are chemically similar and often occur together in nature. The amount of Zn supplement must be balanced, taking into consideration that exposure to high levels of Cd over a long time can lead to kidney failure.
Copper is a metal that occurs naturally in many foods (Table 9), including vegetables, legumes, nuts, grains, fruits, shellfish, avocado, beef, and animal organs (i.e., liver and kidney). Cu shortage in urine could be a cause of seizures (11.3% of DS), since epileptics often exhibit that. However, from the best of our knowledge with DS patients, Cu supplements (especially with Zn intakes) is not recommended since we realised our patients had hypersensitive to Cu and showed more slow growth ratios with Cu-supplement. In a short experiment, we gave DS children (N = 10) 14–28 mg Zn/day and found that all of them had developed Cu deficiency for an unknown reason. Tables 5–7 showed higher Cu concentrations in DS children's whole saliva, parotid saliva, and other biological matrices in comparison with controls. Moreover, we found that Zn-supplementation has decreased the higher level of Cu-DS. For this point, more specific studies shedding light on DS liver health in function to Cu oral supplementation and the inverse relationship between Zn (and maybe Fe) and DS patients' Cu-diet (as Zn/Cu = 2: 1, 5: 1, and 15: 1) should be designed for next study.
Molybdenum is an essential catalyst for enzymes (xanthine oxidase (XO): EC 1.17.3.2, sulphite oxidase: EC 1.8.3.1, and aldehyde oxidase (AO): EC 1.2.3.1) helping to metabolise fats and carbohydrates (CH) and facilitate the breakdown of certain amino acids (AA) in the body. Its role is important to the health. In a short experiment, higher decrease in Mo concentration (despite the soil of the living areas of the patients showed relatively high concentrations of Mo; data not shown) in four DS children (N = 4, 5.63%, three of them have oesophageal cancer family history) was registered which may refer to genetic (genetic sulphite oxidase) deficiency or/and nutritional deficiencies of Mo (Table 9), that could result from the inability to form Mo coenzyme (unknown reason). Those children were suffered from seizures (N = 4/11), opisthotonos (5/11), and lens dislocation (8/11). They have been given ammonium molybdate ((NH4)6Mo7O24) 300 mcg/day IV which caused dramatic recovery, taking the benefit from the Mo's antioxidant properties that helped to break down toxins in the body. However, Cu in biological fluids (i.e., saliva and blood) had decreased when this subgroup of DS patients (N = 4) were given tetrathiomolybdate (H8N2MoS4). By turn, the other DS patients showed normal contents of Mo since enamel contains high amounts of Mo which assisted them in lessening tooth decay. Therefore, it was not exigent to supplement those patients an extra dose of Mo.
Ecological footprint
Fig. 1 proves that food accounts for 15.3% of a household carbon footprint (CF), the higher portion was observed in lower-income households.
The analysis showed that the EF, CF, and CF share of total EF are 5.6 global hectares (gha), 8.8 CO2 tonnes/year, and 54% CO2, respectively. The reduction of these numbers requires direct action to lessen waste and energy use. Thus, to enhance the ecological lifestyle of DS patients, the following recommendations should be addressed:
(1) Governments via city leaders are encouraged to initiate sustainability policies as by adopting urban planning and development strategies to manage the resources depicted in Fig. 1.
(2) Healthcare professionals are encouraged to use telemedicine to reduce the CF of DS healthcare.
(3) Encouraging parents to use renewable energy.
(4) Accessing to family planning by controlling family size to create a sustainable future for those patients, this can largely improve long-term DS footprint.
(5) DS females' rights should be supported and strictly protected.
(6) Controlling diet and cutting food waste which are powerful sustainability levers.
(7) Lessening the amount of meat intake (SI; Ps. 40, 77, and 78) as it is a major source of greenhouse gas emissions (GGEs), along with its other issues as animal welfare, water-use, and land degradation. So that can reduce the DS-EF by enjoin vegetarian-friendly meal days.
(8) Eating more plant-based protein (i.e., beans, legumes, nuts, tofu, and seeds) than animal sources, can cut GGEs. However, these products are rich with phenylalanine (Phe) (Recommended daily limit (RDL): 25 mg/kg body weight or 11 mg/pound) should be controlled.
(9) Eating more fibre-rich foods with low saturated fats (SI; Ps. 36 and 77) and Na, can improve the digestive health, serve in balancing the gut bacteria, and protect against heart disease, colorectal cancer, and diabetes.
(10) Living the green-hipster life. Leafy green as seaweed can be suggested as an environmentally-friendly nutrition for DS.