Serum homocysteine level in newly diagnosed young patients with abnormal glucose tolerance

Abstract

Background:

Hyperhomocysteinemia is an emerging risk factor causing early-onset cardiovascular events. The objective of the study was to assess serum homocysteine levels in newly diagnosed young persons (age < 30 years) with abnormal glucose tolerance.

Methodology:

This cross-sectional study included 40 young participants with newly diagnosed abnormal glucose tolerance (including prediabetes and diabetes mellitus) and an equal number of young persons with normal glucose tolerance (normal glucose tolerance vs prediabetes vs. diabetes mellitus-age (years): 25.0 (22.0, 28.0) vs 25.50 (21.50, 27.0) vs 28.0 (25.0, 29.0), median (interquartile range)). Glycemic status was diagnosed by American Diabetes Association, 2021 criteria. After taking clinical information, fasting blood was collected to measure homocysteine by chemiluminescent immunoassay.

Results:

Homocysteine level was different across the spectrum of glycemic status (normal glucose tolerance vs. prediabetes vs. diabetes mellitus: 15.57 (13.89–20.71) vs 13.19 (11.40–18.11) vs 12.27 (9.97–14.42) µmol/L; median (interquartile range); p = 0.006). Serum homocysteine was significantly elevated in participants with normal glucose tolerance than diabetes mellitus (p = 0.006) but statistically similar in prediabetes in comparison to both normal glucose tolerance and diabetes mellitus (p = NS for both). Homocysteine level was above the normal level in 47.5% of participants with normal glucose tolerance and in 22.5% with abnormal glucose tolerance (p = 0.019).

Conclusions:

Patients under 30 years with diabetes mellitus had lower homocysteine levels than those with normal glucose tolerance.

Introduction

Homocysteine (Hcy) is a sulfhydryl-containing amino acid, an intermediate product in the biosynthesis of the amino acids methionine and cysteine. An elevated level of Hcy is associated with increased cardiovascular risks. Abnormal glucose tolerance (AGT) including prediabetes and diabetes mellitus (DM) is also a major cardiovascular risk factor. Diabetes alone can increase the risk of cardiovascular disease (CVD) by 2–4 fold. ¹ When there is hyperhomocysteinemia in diabetes, CVD risk tends to multiply leading to increased CVD-related morbidity and mortality.^2,3

The global prevalence of diabetes which is 9.3% currently is increasing and is estimated to reach 10.2% by 2030. ⁴ The rising trend of obesity results in an increasing prevalence of type 2 DM (T2DM) at younger ages. ⁵ Currently, the prevalence of impaired fasting glucose and diabetes is high among Bangladeshi children (3.4% and 1.8%, respectively). ⁶ In one study conducted in Bangladesh, among 88 overweight and obese children, metabolic syndrome was present in more than 50% of cases and 60% of the participants had raised insulin resistance measured by homeostatic model assessment (HOMA-IR), and 8% had impaired glucose tolerance. ⁵

South Asian has a “thin-fat phenotype body”’ (muscle thin but body fat), which leads to an increased risk of developing diabetes due to increased abdominal visceral fat and greater insulin resistance in comparison to Europeans. ⁷ It is seen that overweight and obese children are likely to stay obese into adulthood and more likely to develop non-communicable diseases like diabetes and CVDs at a younger age. Traditional risk factors explain only about 60% of the incidences of heart disease. Among other risk factors, hyperhomocysteinemia is one of the emerging risk factors causing cardiovascular disease. ⁸ Early-onset DM (particularly T2DM) is observed to be associated with a higher risk of CVD. ⁹

Hcy may be the prognostic indicator to assess the risk of developing CVD complications, ¹⁰ and it can be a significant predictor of cardiovascular events and death.^11,12 There have been conflicting findings regarding the Hcy level in diabetic patients. According to certain investigations, T2DM patients’ plasma Hcy levels were elevated, maintained, or lowered.^13–15 With this view, we intended to measure serum Hcy in young subjects (<30 years) with AGT.

Methodology

Study design and participants: This cross-sectional study was conducted in the Department of Endocrinology of Bangabandhu Sheikh Mujib Medical University (BSMMU) from July 2020 to October 2021. It included 40 participants of both males and females of age 13–29 years with AGT (including DM and prediabetes) and an equal number of age-matched participants with normal glucose tolerance (NGT). Glycemic status was assigned by oral glucose tolerance test according to American Diabetes Association 2021. ¹⁶ Subjects with known type 1 DM, gestational diabetes mellitus, fibrcalculous pancreatic diabetes (FCPD), secondary diabetes, comorbid diseases (chronic kidney disease, hypothyroidism, psoriasis), and drugs (antiepileptic, methotrexate) that may affect Hcy, acute critical illness, and features of homocystinuria were excluded from the study. The sample size was calculated by using the following formula:

$$\mathrm{n}={({\mathrm{Z}}_{ɑ}+{\mathrm{Z}}_{\beta })}^2\mathrm{X}({\sigma }_1{}^2+{\sigma }_2{}^2)/{({\mu }_1-{\mu }_2)}^2$$

According to a study done by, Bansal et al. ³

μ1 = mean of homocysteine in diabetics = 12.9

σ1 = standard deviation (SD) of homocysteine in diabetics = 5.6

μ2 = mean of homocysteine in non-diabetics = 9.9

σ2 = SD of homocysteine in non-diabetics = 3.6

Z α = 1.96 at 95% confidence level

Z β = 1.28 at 90% power

The calculated sample size is 52 for each group (AGT and NGT). However, the study could include 40 participants in each group.

Study procedure: Assigned participants were requested to visit the site early in the morning after an overnight fast. Clinical information was recorded and a venous blood sample was drawn from the participants. Samples for blood glucose were analyzed on the same day by colorimetric assay by glucose oxidase, whereas samples for homocysteine were allowed to clot and serum was separated by centrifugation and stored at −80°C and later measured by the Atellica IM HCY assay using direct chemiluminescent immunoassay technology using SIEMENS Healthineers Atellica IM Analyzer (Germany). ¹⁷ The intra-assay coefficient of variation for Hcy was less than 7%. Fasting serum levels of Hcy 5–15 µmol/L were considered normal, whereas >15 µmol/L were considered elevated.

Statistical analysis: Data obtained from the study were analyzed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp, Armonk, NY, USA). The data distribution was assessed by the Shapiro–Wilk test. ¹⁸ Results were described in frequencies or percentages for qualitative values and median with interquartile range (IQR) for quantitative values as they had skewed distribution. Pearson’s chi-square and Fisher’s exact test were done for the comparison of qualitative variables across groups, whereas the Kruskal–Wallis one-way ANOVA test was applied for quantitative data with skewed distribution. ¹⁹ p Values ⩽0.05 were considered statistically significant.

Ethical aspects: The study was commenced after approval from the institutional review board of BSMMU. Written informed assent was obtained from the participants or their legally authorized representatives (for participants younger than 18 years of age).

Results

This cross-sectional study included 18 participants with newly diagnosed treatment-naive DM (age: 28.0 (25.0–29.0) years, median (IQR); 61.1% female; hemoglobin A1c (HbA1c) 8.0% (6.6–9.9), median (IQR)), 22 with prediabetes (age: 25.5 (21.5–27.0) years, median (IQR); 59.1% female), and 40 with NGT (age: 25.0 (22.0–28.0) years, median (IQR); 45.0% female). There was no significant difference in respect of body mass index (BMI), waist circumference (WC), waist–hip ratio (WHR), and blood pressure (BP) among the study groups (p > 0.05 for all; Table 1).

Table 1.

Characteristics of the study participants (N = 80).

Variables		NGT (n = 40)	Prediabetes (n = 22)	DM (n = 18)	* p
Age in years, median (IQR)		25.0 (22.0-28.0)	25.5 (21.5-27.0)	28.0 (25.0-29.0)	0.037
Sex	Male	22 (55.0)	9 (40.9)	7 (38.9)	0.436
	Female	18 (45.0)	13 (59.1)	11 (61.1)
BMI	Normal	15 (37.5)	2 (9.1)	3 (16.7)	0.069
	Overweight	6 (15.0)	3 (13.6)	5 (27.8)
	Obese	19 (47.5)	17 (77.3)	10 (55.6)
WC	Normal	14 (35.0)	2 (9.1)	3 (16.7)	0.052
	Centrally obese	26 (65.0)	20 (90.9)	15 (83.3)
WHR	Normal	13 (32.5)	2 (9.1)	4 (22.2)	0.121
	Centrally obese	27 (67.5)	20 (90.9)	14 (77.8)
BP	Normal	21 (52.5)	16 (72.7)	10 (55.6)	0.306
	Elevated	19 (47.5)	6 (27.3)	8 (44.4)
DM in first-degree relatives		21 (52.5)	14 (63.6)	10 (55.6)	0.470

NGT: normal glucose tolerance; DM: diabetes mellitus; BMI: body mass index; WC: waist circumference; WHR: waist–hip ration; BP: blood pressure.

BMI category for age <18 years: normal – 5th to < 85th percentile, overweight – 85th to <95th percentile, obese – ⩾95th percentile; BMI category for age ⩾18 years: normal 18.5–22.9, overweight 23.0–24.9, obese ⩾25 kg/m².

WC category: centrally obese—male > 90 cm, female > 80 cm.

WHR category: centrally obese—male ⩾ 0.90, female ⩾ 0.85.

BP category: elevated—systolic BP⩾130 mmHg and/or diastolic BP ⩾ 80 mmHg.

*

By Pearson’s chi-square test and Kruskal–Wallis one-way ANOVA.

Data were expressed in frequency (%) if not mentioned otherwise, within parentheses are percentages over column total.

Bold indicates significant association.

Hcy level was different across the spectrum of glycemic status (NGT vs prediabetes vs DM: 15.57 (13.89–20.71) vs 13.19 (11.40–18.11) vs 12.27 (9.97–14.42) µmol/L; median (IQR); p = 0.006). Serum Hcy was significantly elevated in participants with NGT than DM (p = 0.006) but statistically similar in prediabetes in comparison to both NGT and DM (p = NS; figure 1). There was an elevation of homocysteine in 47.5% of participants with NGT, whereas only 22.5% of participants with abnormal glucose tolerance (AGT; including both DM and prediabetes) had elevated homocysteine (p = 0.019; Figure 2).

Figure 1.

Serum homocysteine level in various glycemic status.

Figure 2.

Homocysteine category in the study participants (N = 80).

Discussion

Homocysteine is an emerging risk factor for CVD. It has a strong association with metabolic syndrome though studies demonstrating the association of Hcy with diabetes show both positive and negative associations.^20,21 Due to these inconsistent results, the importance of Hcy in the development of micro- or macrovascular disease in diabetes is progressively diminishing. ²² In this cross-sectional observational study, we have tried to see serum homocysteine levels in the population of age group 13–29 years with AGT. The study population had similar BMI, WC, WHR, and BP among different groups. We found that Hcy was significantly elevated in participants with NGT than those with AGT. In the AGT group, around 20% of subjects showed hyperhomocysteinemia; in NGT subjects, it was nearly 50%.

There are various factors that influence serum Hcy levels reported in the literature. The lifestyle determinants that can increase the level of homocysteine in the normal population are low folate intake, smoking, coffee, high-level alcohol consumption, and physical inactivity by altering homocysteine metabolism.^23,24 Homocysteine metabolism also is affected by low vitamin B12 and B6 level, chronic inflammation, and diet. Various studies have shown various levels of serum homocysteine level, that is, higher or similar levels or lower levels in diabetes patients.^{13 -15} These discrepancies may be due to the fact that most studies did not account for folate status and renal function, the two most important determinants of plasma homocysteine level. Decreased plasma homocysteine levels in diabetes and prediabetes patients may be related to stimulatory effect or metabolic interference of insulin and insulin resistance leading to increased remethylation and the hepatic transsulphuration pathway, which is involved in the catabolism pathway of homocysteine.^25,26 Another reason might be due to the increased rate of renal excretion of homocysteine or its metabolites due to hyperfiltration usually present in the early stage of diabetic nephropathy.^27,28

The study had several limitations. First, we could not include an adequate sample to reach desired power. The age of the participants with diabetes was higher than other groups, although the difference was small. Second, the study did not assess folate or vitamin B12 status, which might influence the Hcy level. Finally, the most accepted method for analyzing Hcy is high-performance liquid chromatography or mass spectrometry was not used in the present study. We used the direct chemiluminescent immunoassay method to estimate Hcy, which might also impact the Hcy level.

Conclusion

Despite the fact that hyperhomocysteinemia may be associated with an increased risk of CVD in diabetes, the homocysteine level was found reduced in young Bangladeshi people with diabetes aged less than 30 years compared to those without glucose intolerance.