Analysis of five cases showing false-high Hemoglobin A1c due to reduced catalase activity

Abstract

We encountered five cases that exhibited false-high Hemoglobin A1c (HbA1c) levels when samples were examined using the enzyme-based NORUDIA N HbA1c kit. HbA1c levels were higher than those obtained using other methods, such as HPLC, immune-based methods, and other enzyme-based kits. This kit produced inaccurate results for HbA1c when residual peroxides were present in samples. The addition of peroxidase solution restored false-high HbA1c levels in the five cases, indicating that reduced catalase activity was responsible for these values because catalase eliminates peroxide. Catalase activity and gene mutations were examined in the five cases and an immunohistological analysis was performed to assess the expression of catalase. Cases #1 and 2 were diagnosed as acatalasemia and cases #3, 4, and 5 as hypocatalasemia based on compound heterozygous SNP and heterozygous splicing mutations in the catalase gene. Therefore, impaired catalase activity was responsible for false-high HbA1c levels measured by the NORUDIA N HbA1c kit.

Hemoglobin A1c (HbA1c) is widely used as a biomarker to assess glycemic control and diagnose and treat diabetes mellitus (DM) [1]. HbA1c levels may be affected by various medical conditions, such as anemia and abnormal hemoglobinemia [2]. However, some patients who do not have anemia or abnormal hemoglobinemia may exhibit false-high HbA1c levels when samples are examined using the NORUDIA N HbA1c kit (Sekisui Medical, Tokyo, Japan), which utilizes an enzyme method that may be affected by residual peroxides in samples. Sekisui Medical confirmed that the addition of peroxidase (POD) solution during the measurement process may correct false-high levels; however, the reason for these erroneous values was unclear. We encountered five cases from three different families that exhibited false-high HbA1c levels when samples were examined using the NORUDIA N HbA1c kit. The present study revealed that reduced catalase activity was the cause of these false-high levels.

Materials and Methods

Cases and ethics

Three cases (7, 62, and 78 years old) with false-high HbA1c levels by the NORUDIA N HbA1c kit and the parents (both 37 years old) of a 7-year-old boy were examined in this analysis. The present study was approved by the Ethical Committee on human research at the Dokkyo Medical University Saitama Medical Center (approval number: #22120) and the Ethical Committee of the Graduate School of Medicine, Gifu University (permission number: #29–35) according to the Declaration of Helsinki. Written informed consent was obtained from all participants prior to their enrollment and the use of their genomic DNA and fibroblasts.

Biochemical study

HbA1c levels were measured using the NORUDIA N HbA1c kit. Since information on the POD solution, such as its contents and concentration, is confidential, it was not disclosed by Sekisui Medical. Fasting plasma glucose (FPG) was evaluated using Glucose Auto Stat GA1160^® (Arkray, Kyoto, Japan). Glycoalbumin (GA) was measured using Lucica^®GA-L (Asahi Kasei Pharma Corporation, Tokyo, Japan).

Serum catalase activity assays

Sekisui Medical was in charge of catalase activity measurements and disclosed the value of a healthy subject only as the control. A 20-μL serum sample was used in serum catalase activity assays by the OxiSelect^® Catalase Activity Assay Kit (Cell Biolabs, San Diego, CA, USA).

Catalase gene analysis

Genomic DNA was extracted from the peripheral blood leukocytes of participants using a QIAamp Blood Kit (QIAGEN, Hilden, Germany), and 0.2 μg was used for PCR. We amplified 13 exons and exon-intron border regions by PCR followed by Sanger sequencing. PCR was performed as previously described [3] and PCR primers for all exons were the same as those reported by Kishimoto et al. [4].

Immunohistological assays

Skin fibroblasts were obtained from patients and the healthy control [5, 6], cultured, and then subjected to an immunohistological analysis. The healthy control was a 9-year-old girl whose parents agreed to provide skin fibroblasts. Briefly, cells cultured on a coverslip coated with Geltrex (Thermo Fisher Scientific; #A1048001) were fixed with 4% formaldehyde diluted in phosphate-buffered saline (PBS). After washing with PBS, cells were incubated with a primary antibody at 4°C overnight. Cells were then washed with PBS and incubated with a secondary antibody at room temperature for 2 h or at 4°C overnight. Nuclei were stained with DAPI (1 μg/mL; diluted with PBS; Thermo Fisher Scientific; #D1306), washed with PBS, and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA; #H-1000). Images were captured using an LSM710 confocal laser microscope (Carl-Zeiss). The following antibodies were used: rabbit anti-human catalase (1:500; Athens Research & Technology, Athens, GA, USA; #01-05-030000) and goat anti-rabbit IgG Alexa 488 (1:500; Thermo Fisher Scientific; #A-11008) [6].

Results

Case #2, a 7-year-old boy, underwent adenoidectomy at the Otorhinolaryngology department. After surgery, the oral cavity was sterilized with hydrogen peroxide (H₂O₂), which immediately turned dark brown, indicating acatalasemia. Based on this observation, we hypothesized that reduced catalase activity was the underlying cause of the false-high HbA1c level measured by the NORUDIA N HbA1c kit.

Case #1, a 78-year-old man, was referred to our department in December 2014 with a serum HbA1c level of 8.6%. His blood glucose and GA levels were within normal ranges and he was diagnosed with prediabetes by the 75 g oral glucose tolerance test, which was inconsistent with his HbA1c level and suggested a false-high HbA1c level.

Case #5, a 62-year-old man, was diagnosed with coronary angina pectoris in March 1998 and was treated at our cardiology clinic. A serum HbA1c level of 7.0% was noted in January 2015, and the patient was followed up with diet and exercise therapy.

Since the abnormal absorbance alarm of the NORUDIA N HbA1c kit was activated in February 2017, POD solution was added to a blood specimen. Serum HbA1c levels were 7.6 and 5.7% before and after the addition of the blank solution, respectively, which indicated a false-high HbA1c level due to reduced catalase activity. Therefore, we examined the catalase activity of all participants enrolled in the present study.

We initially measured the HbA1c levels of all participants using several different kits and also assessed FPG and GA (Table 1). As shown in Table 1, HbA1c levels measured using the NORUDIA N HbA1c kit were higher than those measured using other kits. In cases #1, 2, and 5, there were notable inconsistencies between HbA1c levels measured by the NORUDIA N HbA1c kit and FPG or GA. We subsequently added POD solution to the samples of all participants during measurements using the NORUDIA N HbA1c kit, which returned HbA1c levels to within the normal range (Fig. 1).

Table 1

Comparison of HbA1c levels in different measurement kits and FPG and GA levels in the same samples

	method	kit	Case #1 78y/M	Case #2 7y/M	Case #3 37y/M	Case #4 37y/F	Case #5 62y/M
HbA1c (%)	enzyme	NORUDIA N	8.7	6.6	6.1	5.6	6.9
	enzyme	A	6.3	4.9	5.1	5.2	5.4
	enzyme	B	6.2	N/A	N/A	N/A	5.3
	HPLC	C	6.1	N/A	N/A	N/A	5.5
	HPLC	D	6.1	4.9	5.3	5.3	5.5
	immune	E	6.1	4.9	5.2	5.1	5.4
FPG (mg/dL)			114	92	96	91	100
GA (%)			15.4	12.9	12.8	14.5	13.7

Cases #3 and 4 are the father and mother of Case #2, respectively.

N/A: not applicable

Fig. 1

Changes in HbA1c levels measured by the NORUDIA N HbA1c kit upon the addition of POD solution

Upon the addition of H₂O₂ solution to the control blood sample [6, Yamashita, 2017 #12] [5, 6], we observed the formation of foam without any color changes. We added H₂O₂ solution to the blood samples of the participants and observed the loss of foam formation and an evident color change to dark brown in cases #1 and 2. In case #5, we noted reduced foam formation and a mild color change to dark brown.

We also assessed the serum catalase activity of all participants and found the complete loss of catalase activity in cases #1 and 2. In cases #3 and 4, the parents of case #2, and 5, catalase activity was reduced to almost 50% that in the control (Fig. 2).

Fig. 2

Serum catalase activity

A catalase gene analysis revealed two heterozygous single nucleotide polymorphisms (SNPs) (c.-89A/T: rs7943316 and c.-20T/C: rs1049982) in the 5' untranslated region in case #1, indicating compound heterozygous SNPs. These SNPs have been reported in cases of acatalasemia and hypocatalasemia, respectively [7-9]. In case #5, we identified heterozygous splicing variants (IVS4+5g>a: rs762060470) (Supplementary Fig. 1), which were previously reported in a case of acatalasemia [10]. None of the three SNPs were detected in the control.

We obtained skin fibroblasts from case #5 and performed immunostaining for catalase (Fig. 3). An immunopositive catalase level in the fibroblasts of case #5 was detected, but was slightly lower than that in the control [5, 6], suggesting hypocatalasemia, not acatalasemia (Fig. 3). Based on these results, we diagnosed cases #1 and 2 as acatalasemia and cases #3, 4, and 5 as hypocatalasemia (Table 2).

Fig. 3

Catalase (green) expression in cultured skin fibroblasts

Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Table 2

Catalase analysis and diagnoses of 5 cases

	Brownish color change and reduced foaming in blood by H2O2	Catalase analysis			Diagnosis
		Enzyme activity	Protein expression	Gene analysis
Case #1	++	↓↓	N/A	Compound heterologous SNPs in 5' UTR	acatalasemia
Case #2	++	↓↓	N/A	N/A	acatalasemia
Case #3	N/A	↓	N/A	N/A	hypocatalasemia
Case #4	N/A	↓	N/A	N/A	hypocatalasemia
Case #5	+	↓	+	Heterologous splicing variants	hypocatalasemia

N/A: not applicable

Discussion

The NORUDIA N HbA1c kit is used to measure HbA1c levels and the procedure involves pretreating samples to convert hemoglobin to methemoglobin. In the first step, a solution containing protease is added to samples to remove glycosylated dipeptides from the N terminus of the hemoglobin beta chain. Intrinsic catalase in samples removes any peroxides present during this step. In the second step, fructosyl peptide oxidase reacts with the removed glycosylated dipeptides to produce H₂O₂. The amount of H₂O₂ produced affects the intensity of color that develops, and HbA1c levels are assessed by measuring the absorbance of this color. HbA1c levels (%) are then calculated based on the Hb and HbA1c levels obtained in the second step (Fig. 4).

Fig. 4

Schematic figure of the amount of peroxides in blood samples in the reaction process of the NORUDIA N HbA1c kit

However, in the five cases described herein, the peroxide-erasing ability of their samples was low due to the loss of or a reduction in catalase activity. This led to the insufficient erasing of peroxides, resulting in more residual peroxides in the second step and potentially false-high HbA1c levels (Fig. 4).

Enzyme-based measurement kits other than the NORUDIA N HbA1c kit include POD or equivalent enzymes in the first step reagent. As a result, despite having low intrinsic catalase activity, peroxides in samples are effectively removed in the first step, resulting in accurate HbA1c measurements. Catalase is one of the enzymes that removes peroxides in vivo.

In June 2018, Sekisui Medical included a kit insert that included the following statement: “There is a risk of assay results being artificially high in a sub-portion of samples with extremely low POD-like activity.” In November 2018, Sekisui Medical developed the NORUDIA N HbA1c kit, a new HbA1c measurement kit with a lower risk of false-high values.

Acatalasemia was first reported by Takahara in 1949 [11] and is caused by a homozygous mutation in the catalase gene that results in an almost complete catalase deficiency. Acatalasemia is an autosomal recessive disease that is found in consanguineous pedigrees [11]. Hypocatalasemia is a heterozygous form of acatalasemia, in which catalase activity is approximately 50% that of healthy subjects [12].

Although normal bacteria in the oral cavity, such as Streptococcus pneumonia, produce extracellular H₂O₂, H₂O₂ accumulates in the oral cavity of patients with acatalasemia due to catalase deficiency in tissues. When accumulated H₂O₂ interacts with red blood cells, which lack catalase, hemoglobin is oxidized and converted to methemoglobin. Methemoglobin lacks the ability to transport oxygen, resulting in an oxygen-free and undernourished intra-oral cavity that may lead to diseases such as gingival ulcers and gangrene [11]. However, due to improvements in oral hygiene practices and the widespread use of antibiotics, the incidence of oral cavity diseases has decreased, and it is now very rare to encounter acatalasemia in daily practice. The prevalence of acatalasemia and hypocatalasemia are 1/240,000 and 1/600, respectively [13].

Regarding SNPs in the catalase gene, which were detected in the present study, Goth concluded that rs1049982 may not be the causal mutation of acatalasemia because the SNP was observed in healthy subjects [9]. On the other hand, Mansuri et al. reported that catalase activity was significantly reduced in individuals with susceptible genotypes/haplotypes for rs1049982 and rs7943316 polymorphisms [8], which is compatible with case #1.

In conclusion, false-high HbA1c levels measured by the NORUDIA N HbA1c kit appeared to be due to reduced peroxidase-erasing activity caused by catalase deficiency in the five cases. Although acatalasemia is very rare, hypocatalasemia may be encountered in daily practice with a prevalence of 1/600. A previous study demonstrated that the prevalence of DM was high in both acatalasemia and hypocatalasemia [9]. It is important to note that hypocatalasemia is a disease in which HbA1c levels measured by enzyme-based kits, such as the NORUDIA N HbA1c kit, may be falsely elevated in the management of DM.

Data Availability

Data that support the present results are available from the corresponding author upon reasonable request.

Acknowledgments

A part of this work was presented at the 29^th JES Clinical Update on Endocrinology & Metabolism in 2019 and the presentation was published as a proceeding (https://doi.org/10.1507/endocrine.96.S.Update_88) in Japanese in 2020.

This work was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) [grant numbers 23K08015 and 23K10720] (to K.H., K.Hara, respectively) and Dokkyo International Medical Education Research Foundation.

Competing Interests

The authors declare no competing interests.

Disclosure

The authors have nothing to disclose. K.H. is a member of Endocrine Journal’s Editorial Board.

Supplementary Fig. 1

A waveform sequence of IVS4+5g>a

IVS: Intervening sequence

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