Establishment of a reference interval for glycated albumin based on medical check-up data from multiple medical institutions

Abstract Objectives: Data were collected to establish a reference interval for glycated albumin (GA), as well as to calculate a cutoff value for diagnosing diabetes mellitus and the GA level corresponding to a 75-g oral glucose tolerance test (OGTT) 2 h plasma glucose (2h-PG) level of 200 mg/dL. Methods: This study involved 1,843 subjects who were undergoing medical check-ups at several medical institutions and whose HbA1c and GA levels had been measured by OGTT. Results: The GA reference interval that was calculated based on the data obtained from study subjects with normal glucose tolerance was 12.1–17.1%. Using standardized major axis regression, the levels that corresponded to an OGTT 2h-PG level of 11.1 mmol/L were a GA level of 17.5% and an HbA1c level of 47.5 mmol/mol. A receiver-operating characteristic curve analysis was used to calculate the points at which sensitivity and specificity matched as the cutoff values, and the results yielded a GA level of 15.0% (sensitivity 69.3%). Conclusions: The GA reference interval was calculated to be 12.1–17.1%. We propose a GA level of 17.4% as a cutoff value to diagnose diabetes mellitus and a GA level of 15.0% as a screening cutoff value for diabetes mellitus, taking previous reports into account.


Introduction
The prevalence of diabetes mellitus has been increasing rapidly, and it is one of the global health emergencies of the twenty first century.The International Diabetes Federation (IDF) estimated that 537 million people had diabetes mellitus in 2021, and the number is projected to reach 643 million people by 2030.Direct health expenditures due to diabetes mellitus are already close to one trillion USD, and they will exceed this figure by 2030 [1].In 2019, diabetes mellitus was the ninth leading cause of death, with an estimated 1.5 million deaths attributable directly to it [2].In addition, diabetes mellitus often remains undiagnosed.It has been reported that, in 2021, almost one in two (44.7%, or 239.7 million) adults living with diabetes mellitus who were 20 to 79 years of age were unaware of their status, and there are 5 million undiagnosed cases of diabetes mellitus in Japan, accounting for 45.5% of all cases of diabetes mellitus [1].Diabetes mellitus poses a serious threat to global health and has a serious impact on the economy in many countries.
Diabetes mellitus is a disease with few symptoms, which is why blood glucose tests and glycemic control markers are used in screening and as treatment targets.The most basic test is a blood glucose test, but because this test is affected by food, among other factors, markers such as hemoglobin A1c (HbA1c), glycated albumin (GA), and 1,5-anhydroglucitol (1,5-AG) are routinely used for glycemic control because they reflect the average glucose level.Of these, HbA1c indicates a patient's average glucose levels over the past 1 to 2 months, considered the gold standard in diabetes screening, in diagnosis, and when setting treatment targets for patients with diabetes mellitus.However, HbA1c is not perfect, since it does not reflect fluctuations of blood glucose levels, it does not accurately reflect the glycemic control status in subjects with hemoglobinopathy, and falsely low or inaccurate values are obtained depending on the extent of the anemia in hemolytic anemia, in which the red blood cell lifespan is reduced, or in renal anemia [3,4].GA, on the other hand, reflects the average glucose levels over the previous 2-3 weeks, and it is useful for checking treatment efficacy on a monthly basis and when initiating or changing therapy, for example.GA is also not affected by the red blood cell lifespan, and it is becoming internationally widespread as a complement to HbA1c [4].
It is also important to establish reference intervals for glycemic control markers.The reference interval for HbA1c in Japanese people is 26.8-44.3mmol/mol with 42.1 mmol/ mol being the target for achieving normalization of glycemic control status, although a level of 47.5 mmol/mol or above has been established as the recommended reference value for 'Specific Health Checkups' in Japan, and a level of 37.7 mmol/mol or above is the established reference value for 'Specific Health Guidance' in Japan [5].
Fewer data are available on GA than on HbA1c.The Treatment Guide for Diabetes 2019 of the Japan Diabetes Society states that the GA reference interval for Japanese people is 11-16% [5].In a multicenter study that was conducted by the Japan Society of Clinical Chemistry, the reference interval in 434 subjects who underwent a 75-g oral glucose tolerance test (OGTT) was 12.3-16.9%[6].Some information is also available from Europe and North America [7][8][9][10] and China [11].As far as a diabetes diagnostic criterion is concerned, a level of 17.0% was proposed by the Hisayama Study, which was conducted in Japan based on the prevalence of diabetic retinopathy [12].In other countries, studies that were performed using OGTTs in China proposed levels of 17.1% and 16.3% [13,14].In the United States, a report stated that the GA level that corresponds to an HbA1c level of 47.5 mmol/mol is 17.8% [10].Values within a narrow range of 14.5-15.7%have been proposed as GA cutoff values for diabetes screening [15][16][17][18].However, there are no reports of any comprehensive comparisons of the appropriateness of these GA levels.
In the present study, OGTTs were administered to subjects undergoing comprehensive 'Ningen Dock' medical examinations as part of the Japanese health checkup system.A GA reference interval was established, and, among other things, cutoff values for diagnosing diabetes mellitus and the GA level that corresponds to an OGTT 2-h plasma glucose (2h-PG) level of 11.1 mmol/L were analyzed, and the results were compared to those from previous reports.

Study design and study subjects
The present study involved 1,945 subjects who underwent comprehensive 'Ningen Dock' medical examinations including OGTT at Ehime Prefectural Central Hospital, Kinki Central Hospital, Mitsui Memorial Hospital, and Maebashi Red Cross Hospital whose HbA1c and GA levels had been measured.Patients with RBC < 3.3x10 12 /L, hemoglobin < 100 g/L, alanine aminotransferase (ALT) > 80 U/L, aspartate aminotransferase (AST) > 80 U/L, serum albumin < 37 g/L, serum creatinine > 141 µmol/L, urine protein ≥ 1+, or HbA1c < 23.5 mmol/mol were excluded, leaving 1,843 subjects in the study [1,185 men and 658 women; mean age 56.2 ± 9.8 years; mean body mass index (BMI) 23.6 ± 3.2 kg/m 2 ] (Supplementary Table S1).The institutional review board of each study center approved the study protocol, and written, informed consent was obtained from all study subjects.
Diabetes mellitus was diagnosed based on the American Diabetes Association's diagnostic criteria (2003) [19] using the OGTT results or based on study subjects' history of drug therapy for diabetes mellitus.In addition, according to the definition of The Japan Diabetes Society, 20 subjects with a fasting plasma glucose (FPG) level at the time of the OGTT ≥ 7.0 mmol/L or an OGTT 2h-PG ≥ 11.1 mmol/L were classified as diabetic, subjects who were not classified as diabetic were classified as non-diabetic, subjects with FPG < 5.56 mmol/L and OGTT 2h-PG < 7.78 mmol/L were classified as normal, and subjects who were not classified as either diabetic or normal were classified as borderline.

Laboratory methods
Blood samples were obtained after an overnight fast, and laboratory analyses were immediately performed.Blood samples were collected into sodium fluoride-containing blood collection tube, plane tube with coagulation promoted film, and EDTA-2Na-containing blood collection tube for glucose measurement, for measurement of biochemical examination, and for measurement of complete blood counts and HbA1c, respectively.Fasting and post challenge plasma glucose levels were measured by the glucose oxidase method (Adams glucose GA-1153、 Arkray Inc., Kyoto, Japan).HbA1c, expressed as the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) value, was measured by high-performance liquid chromatography using ADAMS-A1c HA-8160 (Arkray Inc., Kyoto, Japan).GA was determined by Hitachi 7600 autoanalyzer (Hitachi Instruments Service Co., Tokyo, Japan), by the enzymatic method using albumin-specific proteinase, ketoamine oxidase, and albumin assay reagent (Lucica GA-L; Asahi Kasei Pharma Co., Tokyo, Japan).Serum concentrations of AST and ALT were measured by Japanese Society of Clinical Chemistry standardization methods (L-type Wako AST and L-type Wako ALT, respectively, Wako Pure Chemical Co., Tokyo, Japan).Serum albumin levels were determined by a modified bromocresol purple method (IatroALB, LSI Medience Co., Tokyo, Japan).Serum concentrations of creatinine were measured using a sarcosine oxidase/peroxidase method (Cygnus Auto CRE, Shino-Test Co., Tokyo, Japan).Serum concentrations of uric acid were measured using a uricase-peroxidase method (Quick Auto Neo UA II、 Shino-Test Co., Tokyo, Japan).Blood cell counts, hematocrit, and hemoglobin were measured by an automated hematology system with a Sysmex SE 9000 (Sysmex Co, Hyogo, Japan).

Statistical analyses
The reference intervals are presented as the medians and the 2.5th and 97.5th percentiles.For the detection of factors affecting the GA and HbA1c levels, stepwise multivariate analysis was performed on the 'normal' group with age, BMI, FPG, OGTT 1h-PG, OGTT 2h-PG, AST, ALT, albumin, uric acid, creatinine, and hemoglobin as explanatory variables.The GA levels that corresponded to the OGTT 2h-PG levels were calculated using standardized major axis regression.The optimal cutoff values for diagnosing diabetes mellitus were calculated by receiver-operating characteristic (ROC) curve analysis.The statistical software used was StatFlex Ver. 7 (StatFlex, Osaka, Japan).

HbA1c and GA distributions
Table 1 shows the HbA1c and GA levels by glucose tolerance for the 1,843 study subjects.The GA reference intervals were 12.1-17.1% and 12.1-17.0%for subjects classified as having normal and non-diabetic glucose tolerance, respectively, and 12.2-17.8%and 12.5-24.0%for subjects classified as having borderline and diabetic glucose tolerance, respectively, indicating that there was considerable overlap with the ranges for subjects classified as having normal glucose tolerance.

Factors affecting normal glucose tolerance
On multivariate analysis including study subjects with normal glucose tolerance with GA, the independent variables were found to be uric acid, BMI, and hemoglobin as significant negative explanatory variables, and creatinine, FPG, and OGTT 1h-PG as significant positive explanatory variables.When a similar investigation was performed for HbA1c, it was confirmed that, as independent variables, hemoglobin was a significant negative explanatory variable, and age, FPG, OGTT 1h-PG, and albumin were significant positive explanatory variables (Supplementary Table S2).

Cutoff values for diabetes screening
An ROC analysis was used to develop cutoff values at which sensitivity and specificity matched, and the results yielded a GA level of 15.0% (sensitivity 69.3%), an HbA1c level of 41.0 mmol/mol (sensitivity 83.1%), an FPG of 5.83 mmol/L (sensitivity 86.3%), and an OGTT 2h-PG of 9.33 mmol/L (sensitivity 93.9%) as cutoff values for diabetes screening (Table 2).

Discussion
In the present study, the following three findings were obtained.First, the GA reference interval in study subjects classified as having normal glucose tolerance was 12.1-17.1%.BMI, uric acid, and hemoglobin had significant negative effects, and creatinine had a positive effect.Second, the GA level corresponding to an OGTT 2h-PG level of 11.1 mmol/L was 17.5%.Third, ROC curve analysis yielded a GA cutoff value for diabetes screening of 15.0%, with sensitivity/specificity of 69.3%.Because there are few reports of such data associated with GA, it was thought that this information would prove to be useful in the future when establishing target values for screening and diagnosing diabetes mellitus and for glycemic control when treating diabetes mellitus.
In the present study, the GA reference interval in Japanese people was 12.1-17.1%.In a multicenter study conducted by the Japan Society of Clinical Chemistry, the reference interval in 434 subjects who underwent an OGTT was reported to be 12.3-16.9%[6].This result is extremely consistent with the results obtained in the present study.Some information is also available from Europe [7], North America [8][9][10], and China (Table 3) [11].According to the Japanese and Chinese studies, the lower limit of the reference interval was distributed from 10.8 to 12.3%, and that of the upper limit was from 16.9 to 17.3%.According to the European and American studies, on the other hand, that of the lower limit was from 9.0 to 11.8%, and that of the upper limit was 15.1-17.8%,both of which are somewhat lower than those of the Japanese and Chinese studies.The results of the present multivariate analysis found that BMI, uric acid, and hemoglobin were independent, negative explanatory variables for the GA level.We have already reported that BMI negatively regulates GA levels in non-diabetic subjects and we proposed that chronic inflammation due to obesity promotes albumin catabolism as a mechanism [12].BMI could have a negative effect on the reference interval of GA, because the Europeans and North American populations tend to have higher BMI levels.However, this issue needs to be studied further in the future.Taking all this into account, it was thought that the GA reference interval of 12.1-17.1% that was obtained in the present study is a valid range.
The reports on GA as a diabetes diagnostic criterion that have been published to date include a report in which the level was calculated to be 17.0% based on the incidence of retinopathy [13], reports in which the level was calculated to be 17.1% and 16.3% based on OGTTs [14,15], and a report from the United States in which a GA level corresponding to an HbA1c level of 47.5 mmol/mol (6.5%) was 17.8% (Table 4) [10].In the present study, a GA level of 17.5% was obtained as a level that corresponds to an OGTT 2h-PG level of 11.1 mmol/L, and a GA level of 17.4% as a level that corresponds to an HbA1c level of 47.5 mmol/mol.As shown in Table 5, though there is some variation among the studies, the mean level, adjusted for the sample size, was 17.4%.It is therefore thought that a GA level of 17.4% is appropriate as a diagnostic criterion for diabetes mellitus.This study has some limitations.First, only Japanese people were included, Second, the age of studied subjects were from 36 to 76 years old and the younger subjects were not included, Third, the average BMI in this study was 23.4 kg/m 2 that was lower compare with Europe and the United States people.
As far as a GA level that could serve as a screening value for diabetes mellitus is concerned, a level of 15.5% was developed in Japan, in which an FPG level of ≥ 7.0 mmol/L or an HbA1c level of ≥ 47.5 mmol/mol is used to diagnose diabetes mellitus [16] and GA levels of 14.5% and 15.0% have been reported in Korea and Taiwan, where diabetes mellitus is diagnosed based on OGTTs (Table 5) [17,18].The results obtained in the present study were very consistent with past reports: diabetes mellitus was diagnosed based on OGTTs, and the calculated GA cutoff value was 15.0%.
A GA reference interval of 12.1-17.1% was therefore established.We would also like to propose a GA level of 17.4% as a cutoff value to diagnose diabetes mellitus and a GA level of 15.0% to screen patients for diabetes mellitus.

Table 4 .
Summary of ga cutoff values for the diagnosis of diabetes mellitus.

Table 5 .
Summary of ga cutoff values for screening for diabetes mellitus.