Measurement in Saliva From Neurotypical Adults of Biomarkers Pertinent to Autism Spectrum Disorders

Figures & data

Figure 1.  Illustration of the comparison of cell counts and concentrations of ten biomarkers that are the most likely potential candidates for use in future studies based on the detection of each analyte in at least 75% of the saliva samples collected (also see Table 5).

Table 1.  Human ELISA kits used.

Variable (Abbreviation)	Kit	Catalog no.	Manufacturer
Dipeptidyl peptidase IV/CD26	Quantikine	DC260	1
IL-12	Quantikine	D1200	1
Total carnitine	Cusabio Biotech Co., Ltd.	CSB-E13242h	2
C4B	USCN Life Science Inc.	E91305Hu	2
GSH	Cusabio Biotech Co., Ltd.	CSB-E09495h	2
MT-2	Cusabio Biotech Co., Ltd.	CSB-E13535h	2
IFN-γ	Quantikine	DIF50	1
GSSG	Cusabio Biotech Co., Ltd.	CSB-E13735h	2

† 1: R&D Systems, Minneapolis MN 55413, USA; 2: Wuhan, Huabei Province 430223, P.R. China.

Table 2.  Comparisons of values detected above lowest possible levels of measurement by ELISA.

Biomarker	Units	n values	MDD	Values >MDD		LLOQ	Values >LLOQ
				n	%		n	%
C4B	ng/ml	24	1.90	24	100	0.50	24	100
CD26	ng/ml	23	0.016	23	100	0.018	23	100
MT-2	ng/ml	23	0.80	23	100	0.19	23	100
GSSG	pg/ml	23	9.38	20	87	0.29	20	87
IFN-γ	pg/ml	22	8.00	13	59	0.90	17	77
Carnitine	nmol/ml	23	0.78	8	35	0.10	11	48
GSH	μg/ml	23	0.20	1	4	0.049	2	9
IL-12	pg/ml	23	5.00	0	0	0.40	2	9

LLOQ: Lower limit of quantitation; MDD: Mean detectable dose.

Table 3.  Analytes for measurement of hormones by MS.

Analyte	Manufacturer	Catalog number
Cortisol	Cerilliant, TX 78665, USA	C-106
Melatonin	Cerilliant, TX 78665, USA	M-095
Serotonin hydrochloride	Sigma Aldrich, MO 63178, USA	H9523
Testosterone	Cerilliant, TX 78665, USA	T-037
N-Acetyl-5-methoxytryptamine-d4	CDN Isotopes, Pointe-Claire, Quebec H9R 1H1, Canada	D-2952
Cortisol-d4	Cerilliant, TX 78665, USA	C-113
Testosterone-d3	Cerilliant, TX 78665, USA	T-046
Human saliva	BioReclamation, NY 11590, USA	HMSALIVA

Table 4.  Hormone analysis by more sensitive methods.

Hormone	Means of analysis	Reagent manufacturer	Limits of detection
Cortisol	Electrochemiluminescent immuno assay	Roche, IN 46256, USA	0.36–30 ng/ml
Melatonin	Radio immuno assay	IBL, MN 55432, USA	1–300 pg/ml
Testosterone	Enzyme linked immunosorbent assay	DRG Diagnostics D-35039 Marburg, Germany	7.1–4500 pg/ml

Table 5.  Biomarker concentrations in saliva from 12 neurotypical adults (six males, six females) sampled at 19:30 and 20:30 h.

Category	Variable	Units	19:30 & 20:30			19:30			20:30			19:30 vs 20:30	19:30 & 20:30			19:30 & 20:30			19:30 & 20:30
			M & F			M & F			M & F			M & F	M			F			M vs F
			n (total)	Mean	±SE	n	Mean	±SE	n	Mean	±SE	t-test p =	n	Mean	±SE	n	Mean	±SE	t-test p =
General	Cells	million	23(23)	2.16	±0.26	11	2.43	±0.44	12	1.92	±0.31	0.345	12	1.90	±0.39	11	2.45	±0.35	0.311
Ubiquitous	Carnitine	nM/ml	11(23)	28.5	±18.1	5	53.9	±38.5	6	7.4	±3.1	0.216	8	37.7	±24.4	3	4.0	±2.2	0.434
	Glutamine	μM/ml	22(23)	10.4	±3.8	11	14.7	±7.5	11	6.1	±1.2	0.270	11	15.4	±7.5	11	5.4	±1.0	0.198
	Glutamic Acid	μM/ml	22(23)	23.3	±5.4	11	28.4	±10.1	11	18.2	±3.7	0.358	11	30.5	±10.2	11	16.1	±2.7	0.185
	GABA	μM/ml	11(23)	2.40	±1.05	5	4.19	±2.11	6	0.91	±0.25	0.122	8	2.80	±1.42	3	1.35	±0.70	0.565
Gastro-intestinal	CD26	ng/ml	23(23)	16.7	±1.8	11	18.1	±3.1	12	15.4	±2.1	0.462	12	21.4	±2.5	11	11.5	±1.6	0.004
Immunologic	C4B	ng/ml	24(24)	198.9	±35.9	12	216.3	±59.4	12	181.5	±42.6	0.639	12	274.2	±63.6	12	123.5	±17.8	0.033
	IFN-g	pg/ml	17(23)	27.0	±4.5	8	27.8	±6.5	9	26.3	±6.6	0.874	10	25.1	±5.4	7	29.7	±8.2	0.629
	IL-12	pg/ml	3(23)	0.53	±0.16	2	0.68	±0.12	1	0.23	–	–	1	0.80	–	2	0.40	±0.17	–
Neurologic	Cortisol	ng/ml	24(24)	1.29	±0.23	12	1.53	±0.43	12	1.06	±0.19	0.327	12	1.77	±0.42	12	0.82	±0.08	0.038
	Melatonin	pg/ml	22(24)	2.59	±0.33	12	2.93	±0.54	10	2.18	±0.31	0.262	10	2.87	±0.61	12	2.36	±0.33	0.450
	Serotonin	ng/ml	2(23)	3.63	±0.39	1	3.24	–	1	4.02	–	–	0	–	–	2	3.63	±0.39	–
	Testosterone	pg/ml	22(23)	31.7	±4.3	11	30.5	±5.8	11	32.9	±6.5	0.781	12	42.0	±5.7	10	1 9.4	±3.8	0.005
Toxicologic	Cysteine	μM/ml	0(23)	–	–	0	–	–	0	–	–	–	0	–	–	0	–	–	–
	GSH	μg/ml	2(23)	2.92	±2.12	2	2.92	±2.12	0	–	–	–	1	0.80	–	1	5.04	–	–
	GSSG	pgl/ml	20(23)	262.7	±17.9	9	295.3	±24.3	11	236.1	±23.8	0.102	11	254.1	±28.5	9	273.2	±20.7	0.610
	MT-2	ng/ml	23(23)	105.5	±13.1	11	100.5	±19.4	12	110.0	±18.5	0.726	12	136.9	±17.3	11	71.2	±14.4	0.009

n(total) = n samples with measurable analyte level determined (n for total samples analyzable); dash (-) if analyte not detected or parameter could not be computed.

Table 6.  Concentration of biomarkers from the current study compared to the literature.

System & biomarker	Current study: Saliva		Literature: Plasma/serum		Age (y [n subjs])	Ref.
	Mean ± SD (units)	Age (y) [n data]	Mean ± SD (units)	Mean (converted units)
Ubiquitous
Carnitine	28.52 ± 59.88 (nmol/ml)	22–80 (24)	51.5 ± 3.3 (μmol/L)	51,500 (nmol/ml)	24–48 (15)	[64]
Glutamine	10.38 ± 18.00 (μM/ml)	22–80 (22)	575 (μmol/L)	84.04 (μM/ml)	ND	[65]
Glutamic acid	23.29 ± 25.16 (μM/ml)	22–80 (22)	54.7 ± 2.16 (μmol/100 ml)	100.4 (μM/ml)	Adult (6)	[66]
GABA	1.18 ± 2.70 (μM/ml)	22–80 (22)	306 ± 79 (pmol/ml)	0.032 (μM/ml)	ND	[67]
Gastrointestinal system
CD26	16.68 ± 8.68 (ng/ml)	22–80 (23)	910 (ng/ml)	910 (ng/ml)	38.5 (100)	[68]
Immunologic system
C4B	198.9 ± 175.0 (ng/ml)	22–80 (24)	62.75^†;124.56^‡ (mg/L)	62,750^†;124,560^‡ (ng/ml)	ND (18)^† (42)^‡	[69]
IFN-γ	26.99 ± 18.58 (pg/ml)	22–80 (22)	139.6 ± 7.9 (pg/ml)	139.6 (pg/ml)	ND (20)	[70]
IL-12	0.533 ± 0.285 (pg/ml)	22–80 (22)	60.7 (pg/ml)	60.7 (pg/ml)	60.8 ± 1.9 (30)	[71]
Neurologic system
Cortisol	1.29 ± 1.14 (ng/ml)	22–80 (24)	250 ± 13 (nmol/L)	90.62 (ng/ml)	19–57 (10)	[72]
Melatonin	2.59 ± 1.54 (pg/ml)	22–80 (22)	0.26 ± 0.13 (nmol/L)	60.39 (pg/ml)	ND (32)	[73]
Serotonin	3.63 ± 0.39 (ng/ml)	24 (2)	0.92 ± 0.17 (μmol/L)	152.12 (ng/ml)	ND (16)	[73]
Testosterone	31.68 ± 20.00 (pg/ml)	23–80 (22)	18.8 ± 0.8 (nmol/L)	5.41 (pg/ml)	19–57 (10)	[72]
Toxicologic system
Cysteine	0 (μM/ml)	NA (NA)	0.95 (mg/100 ml)	9500 (μM/ml)	19–50 (5)	[74]
GSH	3.34 ± 3.35 (μg/ml)	25,80 (2)	2.09 ± 1.14 (μM)	0.642 (μg/ml)	20–43 (59)	[75]
GSSG	262.7 ± 80.1 (pg/ml)	22–80 (20)	0.083 ± 0.090 (μM)	508,504 (pg/ml)	20–43 (59)	[75]
MT-2	105.5 ± 63.0 (ng/ml)	22–80 (23)	3.42 ± 2.30 (ng/ml)	3.42 (ng/ml)	41.3 ± 6.7 (28)	[76]

† One C4B gene (serum).

‡ Two C4B genes (serum).

ND: No data, NA: Not applicable.

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