Saliva nitrite is higher in male children with autism spectrum disorder and positively correlated with serum nitrate

Figures & data

Figure 1. Different pretreatment and sampling methods of saliva. (A) Scenic description of seven pretreatment methods: untreated, centrifugation, saturated ZnSO₄, NaOH plus ZnSO₄ and centrifugation, filtration, centrifugation plus MeOH, and NaOH plus centrifugation. (B) ratio of NO₂⁻ (t_i) to NO₂⁻ (t₀) versus time with methods described in (A). t₀ was when the first measurement was performed immediately after pre-treatment. t_i was when nitrite was measured at time length i: 2, 6, 12 or 24 h. (C) nitrite concentrations within 24 h with one of these two sampling methods: spitting (spit), cotton swabbing (cotton swab), followed by pretreatment of NaOH plus centrifugation.

Table 1. Summary of concentrations of salivary nitrite.

Subjects	Age (years)	No. of subjects	Analytical method	Nitrite (µM)	Ref
ASD children	2–10	126	Chemiluminescence	4.97 ± 3.77 (0.55–16.46)	this work
ND children	2–10	129	Chemiluminescence	2.66 ± 2.07 (0.23–9.83)
ND individuals	–	1	Griess	0–300	[33]
Human	–	6	HPLC	65.1–193.13	[39]
Human	–	4	Ion chromatography	802.9–1475.6	[40]
ND individuals	–	4	Capillary zone electrophoresis	0–30	[36]
Adults	20–50	30	HPLC	204–302	[41]
Human	–	3	Griess	26–176	[34]
ND individuals	–	6	HPLC	92.01 ± 14.11 (75.95–115.878)	[42]
Dental patients	60–84	18	Griess	290.78 ± 56.42	[32]
Adults	19–37	11		125.86 ± 23.87
Gingivitis	33.9 ± 8.38	183	Griess	5.56 ± 4.53 (0.91–16.64)	[31]
Periodontitis	42.65 ± 8.04	150		5.55 ± 5.39 (0–18.28)
Adults	29.9 ± 2.17	147		8.67 ± 8.68 (0.27–33.14)
Overweight or obese	40–65	20	HPLC	62.1 ± 46.3 (13.1–180.1)	[37]
Rampant caries	6–12	30	Griess	32.47 ± 7.00	[45]
ND children	6–12	30		75.04 ± 13.05
Early childhood caries	<6	30	Griess	34.99 ± 5.59
ND children	<6	30		57.45 ± 11.50
Children: DFS < 1	4–6	31	Griess	9.30 ± 5. 96	[38]

Note: ND: normally developing; DFS: diseased and filled surfaces.

DFS: diseased and filled surfaces.

Figure 2. Concentrations of salivary nitrite, serum nitrite and nitrate in the ASD and Control groups. (A) salivary nitrite in the ASD and Control groups (p < 0.0001); (B) serum nitrite in the ASD and Control groups (p < 0.0001); (C) serum nitrate in the ASD and Control groups (p = 0.0109); (D) salivary nitrite of male and female participants in each group; (E) serum nitrite of male and female participants in each group; (F) serum nitrate of male and female participants in each group.

Table 2. Summary of concentrations of nitrite and nitrate in blood among children.

Subjects	Age (years)	No. of subjects	Type	Method	NO2^− (µM)	NO3^− (µM)	Sum (NO2^− + NO3^-) (µM)	Ref
ASD children	2–10	126	serum	Chemiluminescence	0.26 ± 0.20	42.02 ± 23.97	42.28 ± 24.02	this work
ND children	2–10	129	serum	Chemiluminescence	0.18 ± 0.10	35.74 ± 13.49	35.92 ± 13.50
ASD children	–	24	plasma	Griess	N/A	N/A	47.3 ± 5.1/10.8	[19]
ND children	–				N/A	N/A	35.7 ± 4.2/11.8
ASD children	–	27	RBC	Griess	N/A	N/A	^##1.62 ± 0.49	[22]
ND children	–	30			N/A	N/A	^##0.91 ± 0.22
ASD children	–	26	plasma	Griess	N/A	N/A	37.34 ± 6.95	[29]
ND children	–	22			N/A	N/A	27.10 ± 5.76
ADHD	7–11	30	serum	Griess	N/A	N/A	30.3 ± 6.7	[43]
ADHD + ODD	7–11	15			N/A	N/A	36.4 ± 9.8
ND children	7–11	51			N/A	N/A	34.1 ± 8.6
ND individuals	6–15	13	serum	Griess	0.8–5.5	12.8–95.2	N/A	[46]
	16–30	13			0.8–5.0	0.8–76	N/A
	31–45	15			0.8–6.8	2.4–38.4	N/A
	45–60	14			1.0–4.9	0.8–42	N/A
	> 61	14			0.8–3.9	0.8–38.4	N/A

Notes: ##: unit is µmol/g Hb;

ND: normally developing;

ADHD: attention deficit and hyperactivity disorder;

ODD: oppositional defiant disorder.

Figure 3. Correlation among saliva nitrite, serum nitrite and serum nitrate in the ASD group (A–C), in the male participants of the ASD group (D–F), and in the female participants of the ASD group (G–I).

Figure 4. Correlation among saliva nitrite, serum nitrite and serum nitrate in the Control group (A–C), in the male participants of the Control group (D–F), and in the female participants of the Control group (G–I).

Phizackerley PJ, Al-Dabbagh SA. The estimation of nitrate and nitrite in saliva and urine. Anal Biochem. 1983;131(1):242–245.

 PubMed Web of Science ®Google Scholar

Maiti B, Walvekar AP, Krishnamoorthy TS. Separation and determination of inorganic anions by high-performance liquid chromatography using a micellar mobile phase. Analyst. 1989;114:731–733.

 Web of Science ®Google Scholar

Helaleh MI, Korenaga T. Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva. J Chromatogr B Biomed Sci Appl. 2000;744(2):433–437.

 PubMed Web of Science ®Google Scholar

Tanaka Y, Naruishi N, Fukuya H, et al. Simultaneous determination of nitrite, nitrate, thiocyanate and uric acid in human saliva by capillary zone electrophoresis and its application to the study of daily variations. J Chromatogr A. 2004;1051(1–2):193–197.

 PubMed Web of Science ®Google Scholar

Pannala AS, Mani AR, Spencer JP, et al. The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans. Free Radic Biol Med. 2003;34(5):576–584.

 PubMed Web of Science ®Google Scholar

Cardoso TMG, Garcia PT, Coltro WKT. Colorimetric determination of nitrite in clinical, food and environmental samples using microfluidic devices stamped in paper platforms. Anal Methods. 2015;7(17):7311–7317.

 Web of Science ®Google Scholar

Tatarczak-Michalewska M, Flieger J, Kawka J, et al. HPLC-DAD determination of nitrite and nitrate in human saliva utilizing a phosphatidylcholine column. Molecules. 2019;24(9):1754.

 Web of Science ®Google Scholar

Mirvish SS, Reimers KJ, Kutler B, et al. Nitrate and nitrite concentrations in human saliva for men and women at different ages and times of the day and their consistency over time. Eur J Cancer Prev. 2000;9(5):335–342.

 PubMed Web of Science ®Google Scholar

Ali O T, Akalin FA, Sahbazoglu KB, et al. Nitrite and nitrate levels of gingival crevicular fluid and saliva in subjects with gingivitis and chronic periodontitis. J Oral Maxillofac Res. 2014;5(2):e5.

 Google Scholar

Modi A, Morou-Bermudez E, Vergara J, et al. Validation of two point-of-care tests against standard lab measures of NO in saliva and in serum. Nitric Oxide. 2017;64:16–21.

 PubMed Web of Science ®Google Scholar

Hegde AM, Neekhra V, Shetty S. Evaluation of levels of nitric oxide in saliva of children with rampant caries and early childhood caries: a comparative study. J Clin Pediatr Dent. 2008;32(4):283–286.

 PubMed Web of Science ®Google Scholar

Ghasempour M, Qujeq D, Rabiee M, et al. Measurement of nitrite and nitrate in saliva of children with different caries activity. J Contemp Dent Pract. 2014;15(5):623–625.

 PubMedGoogle Scholar

Tostes MH, Teixeira HC, Gattaz WF, et al. Altered neurotrophin, neuropeptide, cytokines and nitric oxide levels in autism. Pharmacopsychiatry. 2012;45(6):241–243.

 PubMed Web of Science ®Google Scholar

Al-Mashhadi A, Checa A, Wåhlin N, et al. Changes in arterial pressure and markers of nitric oxide homeostasis and oxidative stress following surgical correction of hydronephrosis in children. Pediatr Nephrol. 2018;33(4):639–649.

 PubMed Web of Science ®Google Scholar

Zoroğlu SS, Yürekli M, Meram I, et al. Pathophysiological role of nitric oxide and adrenomedullin in autism. Cell Biochem Funct. 2003;21(1):55–60.

 PubMed Web of Science ®Google Scholar

Tas FV, Guvenir T, Tas G, et al. Nitric oxide levels in disruptive behavioral disorder. Neuropsychobiology. 2006;53(4):176–180.

 PubMed Web of Science ®Google Scholar

Toprakçi M, Ozmen D, Mutaf I, et al. Age-associated changes in nitric oxide metabolites nitrite and nitrate. Int J Clin Lab Res. 2000;30(2):83–85.

 PubMedGoogle Scholar

Data availability statement

Due to the nature of this research, participants of this study did not agree for their personal data to be shared publicly, and they agreed to share the measured biochemical data online. All the biochemical data used in this study have been deposited to the https://data.mendeley.com/ database (DOI:10.17632/8kz7rwmwnt.1).