Salivary 1,5-AG: A Non-Invasive Biomarker for Monitoring Diabetes mellitus T2DM

Diagnostic Ability of 1,5 AG in Serum

1,5 AG possesses several advantageous properties as a glycemic biomarker.

• It is not affected by the time of sample collection and demonstrates minimal sensitivity to sampling conditions such as hemolysis, use of serum versus plasma, types of reagents present in the sampling tube, or delays in sample processing.
• Unlike HbA1c, 1,5 AG demonstrates greater sensitivity to short-term glycemic fluctuations, particularly in the 6–11% HbA1c range, and is less prone to analytical variability (Yamanouchi 5, Akanuma et al. 1991).
• Multiple studies have reported substantial variability in 1,5 AG levels, with similar HbA1c values, making it valuable for detecting glycemic excursions.
• In individuals with well or moderately controlled diabetes, 1,5 AG exhibits a stronger correlation with glucose variability than HbA1c or FA.
• The distribution of 1,5 AG values is systematic and spans a broad range corresponding to varying degrees of glycemic control. This wide range enhances its discriminatory capacity, making it more effective than traditional markers in identifying clinically relevant differences in glycemic status. Previous studies have underscored the advantages of serum 1,5 AG over conventional glycemic markers such as HbA1c and fructosamine, particularly in detecting glycemic fluctuations within near-normal ranges, owing to its greater variability.
• Under well-controlled glycemic conditions, the daily increment of plasma 1,5 AG remains consistent at approximately 0.296 ± 0.18 µg/mL across individuals, independent of treatment modality, sex, age, body weight, or diabetes duration. Moreover, a strong inverse correlation has been observed between the reduction in plasma 1,5-AG and urinary glucose excretion. Compared to traditional tests, 1,5 AG offers distinct advantages as a sensitive, detailed, and precise marker for glycemic control in diabetes.
• 1,5 AG possesses predictive utility; glycemic status can be assessed using the A · G index (1,5 AG x Urinary Glucose = 16), enabling estimation of urinary glucose levels based on plasma 1,5 AG concentration. Its measurement is not influenced by the time of the day, enhancing its clinical applicability.
• When it comes to dysglycemia identification, 1,5 AG exhibits a modest diagnostic accuracy. Good and poor glycemic control can often be distinguished by an ideal cutoff in the range of 14–16 µg/mL. With 69% sensitivity and 72% specificity (AUC~0.78), a study in high-risk individuals found 15.9µg/mL to be the optimal cut-off for diabetes screening. Likewise, in another cohort, 1,5AG <92>

1,5 AG and HbA1c

Glycated hemoglobin, HbA1c, remains the gold standard for evaluating long-term glycemic control in the management of diabetes and is a well-established predictor of diabetes-related complications. However, its reliability may be compromised by several limitations. HbA1c levels are influenced by the lifespan of red blood cells and tend to disproportionately reflect glucose exposure from the preceding 30 days, thereby failing to capture recent acute fluctuations in the blood glucose levels. Intra- and inter-individual variability, particularly at lower HbA1c levels, further complicates interpretations. Moreover, HbA1c does not differentiate between patients achieving glycemic targets through glycemic variability (i.e., alternating episodes of hypo- and hyperglycemia) and those maintaining consistently stable glucose levels. It also lacks the ability to distinguish between postprandial and fasting hyperglycemia, thereby limiting its clinical utility in guiding treatment strategies, especially in patients with moderately controlled diabetes (Dungan 2008). Additionally, differences in assay methodologies contribute to significant interlaboratory variability in HbA1c measurements. Individual variability in glycation and deglycation around the normal range of HbA1c further affects accuracy. Integrating HbA1c with complementary markers such as 1,5 AG may enhance the precision of glycemic assessment and support more effective diabetes management (Yamanouchi 2, Ogata et al. 1996).

Combining 1,5-AG with HbA1c will improve the detection of short-term glycemic decline, although HbA1c remains the gold-standard marker for diabetes diagnosis and chronic glycemic control. Using 1,5-AG together with HbA1c enhances the diagnostic performance. Yamanouchi et al. (1997) showed that for impaired glucose tolerance (IGT), 1,5-AG and HbA1c taken together provided better screening. Comparably, in a study by Ying, He et al., stepwise regression analysis found HbA1c and 2-hour plasma glucose (2hPG) as independent predictors of 1,5-AG levels (β = -0.295 and -0.298, respectively; p<0>

The different physiological processes behind these markers help to explain their complementary diagnostic power. HbA1c shows chronic glycemia while 1,5-AG is affected by transient hyperglycemia and renal glycosuria. In evaluating post-prandial glucose levels in those moderate-control patients (HbA1c <8> A low 1,5 Anhydroglucitol HemoglobinA1c Index (AHI) (indicating low 1,5-AG relative to HbA1c) correlates with worse glycemic variability and β-cell activity; some researchers have proposed an integrated metric, the AH Index (AHI = 1,5-AG × HbA1c / 100), to jointly evaluate long-term and short-term control. These results highlight how adding 1,5-AG offers diagnostic insight into short-term fluctuations and acute risk that HbA1c may overlook, even while average control is gauged by it.

1,5AG and FPG

Combining 1,5-AG with fasting plasma glucose (FPG) greatly increases diagnostic performance for diabetes screening. Besides improving AUC to 0.912, adding 1,5-AG to the FPG criterion raised the screening sensitivity from 69% (1,5-AG alone) to 82.5%, and specificity from 72% to 83.5%, in a cohort (N=3098) (Ying, He et al. 2017). FPG and 1,5-AG capture complementary features of glycemia. Whereas 1,5-AG shows postprandial spikes, FPG reflects basal glucose. When used together, both markers help to identify cases of isolated postprandial hyperglycemia, normal FPG but high 2-hour glucose. Indeed, screening based on FPG alone would miss many people with isolated post-prandial hyperglycemia without 1,5-AG. The study revealed that using FPG+1,5-AG criteria allowed 75% of subjects to avoid an OGTT, 44?wer OGTTs than using FPG cutoffs alone; the combined approach can significantly lower the need for oral glucose tolerance tests. By identifying post-prandial dysglycemia (Ying, He et al. 2017), 1,5-AG combined with FPG boosts diagnostic efficiency.

Salivary 1,5 Ag in Glucose Monitoring

Saliva is a clinically valuable biofluid with significant potential for screening, diagnosis, and patient management in both oral and systemic diseases. Rich in diverse biomarkers, it supports the development of multiplexed assays for use in point-of-care devices and rapid diagnostic tests. Saliva biomarkers are regarded as functional equivalents to serum biomarkers, as they partially mirror the physiological and pathological state of the body. Of 94 metabolites that were associated with T2DM, only three are detectable in saliva, 1,5 AG being one of them.

A population-based screening study using LC-MS has shown that saliva 1,5-AG levels were notably lower in persons with diabetes. This study further established a saliva 1,5-AG cutoff of 0.44 µg/mL for diabetes detection, thus improving diagnostic efficiency when 1,5 AG is used with HbA1c or fasting plasma glucose. Covariates such as age, gender, BMI, and ethnicity does not significantly influence 1,5-AG levels. Notably, samples collected in a nonfasting state showed a strong association with T2D, highlighting the robustness of salivary 1,5-AG as a reliable, non-invasive, and practical screening tool for diabetes, even under nonfasting conditions .

 Research Gap

Despite significant advances in salivary diagnostics and the growing recognition of saliva as a viable medium for systemic disease detection, the clinical utility of salivary 1,5-anhydroglucitol (1,5-AG) as a non-invasive biomarker for glycemic control remains underexplored. While 1,5-AG has demonstrated superior sensitivity to glycemic excursions compared to HbA1c and fructosamine in serum, its application in saliva is limited by methodological inconsistencies, particularly with enzymatic assays that may overestimate concentrations due to interference from structurally similar sugars. Moreover, although early detection of type 2 diabetes mellitus (T2DM) is essential for preventing complications and improving quality-adjusted life years (QALYs), current diagnostic approaches rely on invasive blood sampling, which may not be feasible or scalable in resource-limited or high-anxiety populations.

Preliminary Data

Methods

Unstimulated whole saliva samples were collected from 42 adult participants, including 21 individuals with Type 2 Diabetes Mellitus (T2DM) and 21 age- and sex-matched healthy controls. All samples were collected in the morning following an overnight fast, under standardized conditions to minimize circadian and dietary influences.

Salivary 1,5-anhydroglucitol (1,5-AG) levels were quantified using a commercially available colorimetric ELISA kit (Abcam, Cambridge, UK), according to the manufacturer’s instructions. Samples were analyzed in duplicate, both spiked and unspiked, to account for potential matrix effects associated with low analyte concentrations in saliva.

For spiked samples, 2 µL of 500 µM 1,5-AG standard (1 nmol) was added to the same volume of sample, and the total volume was adjusted to 30 µL with Sample Pretreatment Buffer. A standard curve ranging from 0 to 5 nmol was generated using serial dilutions of the working 500 µM standard solution. All wells received 20 µL of Pretreatment Reaction Mix and were incubated at 37°C for 90 minutes. Subsequently, 50 µL of Detection Reaction Mix was added to each well, followed by incubation at 37°C for an additional 60 minutes in the dark.

Absorbance was measured at 460 nm using a microplate reader in endpoint mode (BioTek Synergy HTX, USA). Analyte concentrations were calculated from the standard curve after blank correction. Spike-in recovery was used to validate measurement accuracy. Final concentrations were expressed in mM, accounting for sample volume and any dilution factors.

All statistical analyses were performed in R (version 4.3.2). To assess the distribution of salivary 1,5-AG concentrations, the Shapiro–Wilk test was applied separately to the healthy and Type 2 Diabetes Mellitus groups. As at least one group deviated from normality, group comparisons were conducted using the non-parametric Mann–Whitney U test (Wilcoxon rank-sum test). To examine associations between salivary 1,5-AG and clinical glycemic parameters, simple linear regression analyses were performed with fasting plasma glucose (FPG) and HbA1c as independent variables. A p-value of <0>

Table 1. Demographic and clinical characteristics of participants (n=42)