Tissue AGE Levels Research Articles

The association of coronary artery disease and advanced glycation end-products

Traditional risk factors are insufficient to explain all cases of Coronary Artery Disease (CAD) in patients with Diabetes Mellitus (DM). Hyperglycemia is the hallmark feature of DM. An increase in the incidence of both micro-and macrovascular complications of diabetes has been observed with increased duration of hyperglycemia. This association persists even after glycemic control has been achieved, suggesting an innate mechanism of “metabolic memory.” Advanced Glycation End products (AGEs) are glycated proteins or lipoproteins that may serve as mediators of metabolic memory due to their increased production in the setting of hyperglycemia and generally slow turnover. Elevated AGE levels can lead to abnormal cross linking of extracellular and intracellular proteins disrupting their normal structure and function. Furthermore, activation of AGE receptors can induce complex signaling pathways leading to increased inflammation, oxidative stress, enhanced calcium deposition, and increased vascular smooth muscle apoptosis, contributing to the development of atherosclerosis. Through these mechanisms, AGEs and their receptors may play important roles in the development and progression of CAD. However, clinical studies regarding the role of AGEs and their receptors in advancing CAD are limited, with contradictory results. Further studies are needed to evaluate the utility of circulating and tissue AGE levels in identifying asymptomatic patients at risk for CAD or to identify patients who may benefit from invasive intervention.

Inhibitors of Advanced Glycation End Product (AGE) Formation and Accumulation.

A range of chemically different compounds are known to inhibit the formation and accumulation of advanced glycation end products (AGEs) or disrupt associated signalling pathways. There is evidence that some of these agents can provide end-organ protection in chronic diseases including diabetes. Whilst this group of therapeutics are structurally and functionally different and have a range of mechanisms of action, they ultimately reduce the deleterious actions and the tissue burden of advanced glycation end products. To date it remains unclear if this is due to the reduction in tissue AGE levels per se or the modulation of downstream signal pathways. Some of these agents either stimulate antioxidant defence or reduce the formation of reactive oxygen species (ROS), modify lipid profiles and inhibit inflammation. A number of existing treatments for glucose lowering, hypertension and hyperlipidaemia are also known to reduce AGE formation as a by-product of their action. Targeted AGE formation inhibitors or AGE cross-link breakers have been developed and have shown beneficial effects in animal models of diabetic complications as well as other chronic conditions. However, only a few of these agents have progressed to clinical development. The failure of clinical translation highlights the importance of further investigation of the advanced glycation pathway, the diverse actions of agents which interfere with AGE formation, cross-linking or AGE receptor activation and their effect on the development and progression of chronic diseases including diabetic complications. Advanced glycation end products (AGEs) are (1) proteins or lipids that become glycated as a result of exposure to sugars or (2) non-proteinaceous oxidised lipids. They are implicated in ageing and the development, or worsening, of many degenerative diseases, such as diabetes, atherosclerosis, chronic kidney and Alzheimer's disease. Several antihypertensive and antidiabetic agents and statins also indirectly lower AGEs. Direct AGE inhibitors currently investigated include pyridoxamine and epalrestat, the inhibition of the formation of reactive dicarbonyls such as methylglyoxal as an important precursor of AGEs via increased activation of the detoxifying enzyme Glo-1 and inhibitors of NOX-derived ROS to reduce the AGE/RAGE signalling.

Skin fluorescence as a clinical tool for non-invasive assessment of advanced glycation and long-term complications of diabetes.

Glycation is important in the development of complications of diabetes mellitus and may have a central role in the well-described glycaemic memory effect in developing these complications. Skin fluorescence has emerged over the last decade as a non-invasive method for assessing accumulation of advanced glycation endproducts. Skin fluorescence is independently related to micro- and macrovascular complications in both type 1 and type 2 diabetes mellitus and is associated with mortality in type 2 diabetes. The relation between skin fluorescence and cardiovascular disease also extends to other conditions with increased tissue AGE levels, such as renal failure. Besides cardiovascular complications, skin fluorescence has been associated, more recently, with other prevalent conditions in diabetes, such as brain atrophy and depression. Furthermore, skin fluorescence is related to past long-term glycaemic control and clinical markers of cardiovascular disease. This review will discuss the technique of skin fluorescence, its validation as a marker of tissue AGE accumulation, and its use as a clinical tool for the prediction of long-term complications in diabetes mellitus.

Role of Reactive Oxygen Species and Advanced Glycation End Products in the Malfunctioning of Dental Implants.

In the last decade, dental implants have emerged as a crucial modality and serve as an individual form of therapy for dental failure. However, disparities in host responses have led to peri-implantitis and implant failure. The pathological mechanisms driving peri-implantitis remain largely unknown. In this study, we evaluated the role of oxidative stress and advanced glycation end products (AGE) in the progression of peri-implantitis and dental implants failure, compared with chronic periodontal disease. Three patient groups (peri-implantitis, chronic periodontal disease and control), each with 10 subjects (7M/3F) and average age ranging from 40-60 years were selected for analysis. Salivary oxidative stress and tissue AGE levels were analysed by probing for reactive oxygen species (ROS) and Maillard reaction-related fluorescence, respectively. We observed significant increase (> 2-fold) in oxidative stress and AGE levels in patients with peri-implantitis and chronic periodontal disease compared to controls, with chronic periodontal disease having the highest levels. In addition, we observed a strong positive correlation (r = 0.94) between oxidative stress and AGE levels in the patients. We propose that increased AGE levels and oxidative stress, although not the only pathway, are significant mediators in the pathogenesis of peri-implantitis. Altering them may potentially be used in combination with other modalities to manage peri-implantitis.

Dietary glycotoxins induce RAGE and VEGF up-regulation in the retina of normal rats

Exogenous intake of glycotoxins present in western diet accelerates the accumulation of advanced glycation end products (AGEs) in multiple organs leading to potential tissue damage. Advanced ageing and diabetic conditions have been associated with AGEs deposition in multiple eye compartments including Bruch's membrane, optic nerve, lens and cornea. However, the impact of dietary AGEs in ocular physiology has not been extensively studied. The present study investigates the direct effects of a high AGE content diet in the ocular tissues of normal rats of different age. Two groups of baby (4 weeks of age) and adult (12 weeks of age) female Wistar rats (n = 73) were allocated to high- or low-AGE diet for 3 months. Upon completion of experimental protocol, somatometric, hormonal and biochemical parameters were evaluated in all groups. Circulating and tissue AGE levels were estimated along with their signaling receptor (receptor for AGEs, RAGE) and vascular endothelial growth factor A (VEGF-A) expression in ocular tissues of the different subgroups. High AGE intake was associated with elevated serum AGEs (p = 0.0001), fructosamine (p = 0.0004) and CRP levels (p = 0.0001) compared to low AGE. High peripheral AGE levels were positively correlated with significant increased tissue immunoreactivity of AGEs and RAGE in retinal and uveal tissues as well as retinal VEGF-A expression. Up-regulation of RAGE and VEGF-A expression was observed in the ocular tissue of both baby and adult animals fed with high-AGE diet. Co-localization of AGEs and RAGE staining was observed mainly in the inner retinal layers and the retinal pigment epithelium (RPE) of all groups. VEGF-A expression was elevated in the RPE, the inner nuclear layer and the retinal ganglion cell layer of the animals exposed to high-AGE diet. In conclusion, dietary AGEs intake affects the physiology of ocular tissues by up-regulating RAGE and VEGF-A expression contributing to enhanced inflammatory responses and pathologic neovascularization in normal organisms independent of ageing.

Type 2 diabetes, skin autofluorescence, and brain atrophy.

Type 2 diabetes mellitus (T2DM) is associated with brain atrophy, but the mechanisms underlying this link are unknown. Advanced glycation end products (AGEs) accumulate in T2DM, resulting in inflammation, oxidative stress, and protein cross-linking, which are known contributors to neurodegeneration. We aimed to study whether tissue AGE accumulation is associated with T2DM-related brain atrophy. We performed brain magnetic resonance imaging, cognitive tests, and noninvasive skin autofluorescence (SAF; a measure of tissue AGE levels) on people aged >55 years with and without T2DM. Multivariable linear regression was used to study the relationships among T2DM, SAF, and gray matter volume (GMV). There were 486 people included in the study. T2DM was associated with greater SAF. Greater SAF, T2DM, and cognitive impairment were each associated with lower GMV independently of age, sex, and total intracranial volume. SAF partially mediated the association between T2DM and GMV. Longitudinal studies may help confirm whether tissue AGE accumulation is associated with brain atrophy in T2DM.

Decreased serum carnitine is independently correlated with increased tissue accumulation levels of advanced glycation end products in haemodialysis patients

There is accumulating evidence that advanced glycation end products (AGE) play a role in cardiovascular disease (CVD) in patients with haemodialysis (HD). Carnitine deficiency is frequently observed in HD patients, which may also contribute to CVD. In this study, we examined whether carnitine deficiency was independently associated with increased tissue accumulation levels of AGE in HD patients. One hundred and twenty-nine HD patients underwent determinations of blood chemistries including serum level of carnitine. Tissue AGE levels were evaluated by measuring skin autofluorescence with an AGE-reader. Serum carnitine levels were significantly lower, while skin AGE levels were significantly higher in HD patients compared with healthy controls (P < 0.001). In univariate analysis, β(2)-microglobulin (β(2)-MG) and carnitine (inversely) were correlated with skin AGE levels. Multiple stepwise regression analysis revealed that carnitine levels were one of the independent determinants of skin AGE levels (P = 0.024). When β(2)-MG-adjusted skin AGE levels were stratified by serum carnitine levels, a statistical significance and dose-response relationship were observed (P = 0.043). Furthermore, skin AGE levels were one of the independent determinants of serum carnitine levels as well (P = 0.012). The present study demonstrated that decreased carnitine levels were independently associated with increased skin AGE levels in HD patients. Since carnitine is reported to inhibit the formation of AGE in vitro, our study suggests that supplementation of carnitine may be a therapeutic target for preventing the accumulation of tissue AGE and subsequently reducing the risk of CVD in HD patients.

Tissue level of advanced glycation end products is an independent determinant of high‐sensitivity C‐reactive protein levels in haemodialysis patients

C-reactive protein (CRP) level predicts future cardiovascular events in patients on haemodialysis (HD). Advanced glycation end products (AGE) play a role in cardiovascular disease (CVD) in HD patients. However, which variables including tissue AGE levels are independently associated with CRP remains unknown. Therefore, whether tissue AGE and CRP levels were correlated with atherosclerosis in HD patients was examined. Fifty-four HD patients underwent determinations of blood chemistries and tissue AGE. Tissue AGE levels were evaluated by measuring skin autofluorescence. Pulsatility index (PI) in the carotid artery was measured using a Doppler ultrasonography. Univariate analyses showed that age, white blood cells, serum albumin (inversely), alkaline phosphatase (inversely), tartrate-resistant acid phosphatase 5b (TRAP5b) (inversely) and skin AGE levels were significantly correlated with high-sensitivity CRP (hsCRP). Multiple stepwise regression analysis revealed that serum albumin, TRAP5b and skin AGE levels were independent determinants of hsCRP. Further, PI was highest among HD patients with high skin AGE and high hsCRP levels. The present study suggests that tissue AGE level is one of the independent determinants of hsCRP in HD patients. Tissue AGE and hsCRP levels may be correlated with each other, which could in concert contribute to the progression of atherosclerosis in these subjects.

Tissue advanced glycation end products are associated with diastolic function and aerobic exercise capacity in diabetic heart failure patients

Advanced glycation end products (AGEs) are increased in patients with diabetes and are associated with diastolic dysfunction through the formation of collagen crosslinks in the heart. The association among AGEs, diastolic function, and aerobic capacity in heart failure (HF) patients with and without diabetes is, however, unknown. We therefore studied the association among tissue AGEs, diastolic function, and aerobic capacity in patients with HF with or without diabetes. In chronic HF patients (with and without left ventricular systolic dysfunction), tissue AGEs [skin autofluorescence (AF)], diastolic function (echocardiographic mean E' and E/E'), and aerobic capacity [peak oxygen uptake (VO(2)) on cardiopulmonary exercise testing] were obtained. A total of 49 diabetics and 156 non-diabetics were included. Diabetics were older and had more cardiovascular risk factors, but left ventricular ejection fractions (LVEF) were similar. Tissue AGEs were higher in diabetics compared with non-diabetics (2.8 ± 0.8 vs. 2.3 ± 0.7 a.u.; P < 0.001). Furthermore, there was a correlation between tissue AGEs and mean E' (r = -0.30; P < 0.001, after adjustment for age, r = -0.21; P = 0.004). Aerobic capacity was significantly lower in diabetic patients with HF (peak VO(2): 17.4 ± 5.1 vs. 21.7 ± 6.1 mL/min/kg; P = 0.001), even after adjustment for age and LVEF. Peak VO(2) was related to skin AF (P = 0.03), independent of age, diabetes, LVEF, and New York Heart Association functional class. Patients with diabetes and HF have similar LVEF but poorer exercise capacity compared with non-diabetic HF patients. Our data suggest that these findings might be explained by the observed association among tissue AGE levels, diastolic function, and exercise capacity.

Tissue-Advanced Glycation End Product Concentration in Dialysis Patients

Tissue-advanced glycation end products (AGE) are a measure of cumulative metabolic stress. Assessment of tissue AGE by skin autofluoresence (AF) correlates well with cardiovascular outcomes in hemodialysis (HD) patients. This study aimed to measure and compare tissue AGE levels in HD and peritoneal dialysis (PD) patients and to evaluate the impact of systemic PD glucose exposure. Tissue AGE were measured in 115 established dialysis patients (62 HD and 53 PD) using a cutaneous AF device (AGE Reader; DiagnOptics). Values were compared with an age-matched non-chronic kidney disease database. Review of all previous PD solution delivery/prescription data determined PD glucose exposure. PD patients were similar in age to HD patients but had a shorter dialysis vintage. There were no differences in ischemic heart disease or smoking history, statin or angiotensin-converting enzyme inhibitor (ACEi) use, lipids, biochemistry, or prevalence of diabetes. More than 90% of both groups had met current dialysis adequacy targets. Skin AF values in PD and HD patients were similar and strongly correlated with historical PD glucose exposure. Skin AF correlated with age in both groups but with dialysis vintage only in PD patients Cumulative metabolic stress and transient hyperglycemia results in grossly elevated levels of tissue AGE in dialysis patients. In PD patients, this high level of AGE deposition is associated with historical glucose exposure. This observation provides a previously unappreciated potential link between PD exposure to glucose and systemic cardiovascular disease.

Possible link of food-derived advanced glycation end products (AGEs) to the development of diabetes

China Da Qing diabetes prevention study has recently shown that group-based lifestyle interventions over six years can prevent or delay the development of diabetes in patients with impaired glucose tolerance (IGT) for up to 14 years after the active intervention. These findings suggest the sustained beneficial effects of lifestyle interventions to prevent diabetes in at-risk patients for diabetes. Therefore, the clinical study suggests that so-called 'metabolic memory' is involved in the development of diabetes. Potential mechanisms for propagating this 'metabolic memory' are the non-enzymatic glycation of proteins. The formation and accumulation of advanced glycation end products (AGEs) have been known to progress at an accelerated rate under diabetes, and there is accumulating evidence that AGEs play a role in the development of diabetes by inducing islet beta cell damage and/or insulin resistance. Further, there are several animal studies to suggest that dietary AGEs are involved in insulin resistance, visceral obesity and the development of diabetes. These findings led us to speculate that the long-term beneficial influence of early lifestyle interventions on the development of diabetes could be ascribed, at least in part, to their inhibitory effects on AGEs. That is, intake of food-derived AGEs may be suppressed in the lifestyle intervention group, which could reduce the risk for the development of diabetes in high-risk patients with IGT. Therefore, it is an interesting issue to clarify whether food-derived AGEs are actually restricted during the active intervention period and if circulating or tissue AGE levels at the closure of the China Da Qing diabetes prevention study could predict the risk for the development of diabetes 14 years after the trial. This additional, clinical investigation may provide us more information about whether restriction of food-derived AGEs is beneficial for the prevention of the development of diabetes in high-risk patients and may be a novel therapeutic target to prevent diabetes and its related complications.

Therapeutic Interruption of Advanced Glycation in Diabetic Nephropathy

A major common feature of the chemically disparate compounds that inhibit advanced glycation end product (AGE) accumulation or signaling is their ability to show end-organ protection in experimental models of diabetes complications. The mechanisms by which these AGE- lowering therapies confer their benefits remain unsolved. Is it the reduction in tissue AGE levels per se or the inhibition of downstream signal transduction (as has been described with the soluble receptor for AGE)? Possible modes of action that need to be investigated include the ability of some of these agents to stimulate antioxidant defenses, to lower cholesterol and other lipid levels, and to inhibit low-grade inflammation. To understand these novel mechanisms, further examination of the advanced glycation pathway and, in particular, the diverse action of these agents in ameliorating the development of diabetic complications is needed.

The Advanced Glycation End Product N ε-Carboxymethyllysine Is Not a Predictor of Cardiovascular Events and Renal Outcomes in Patients With Type 2 Diabetic Kidney Disease and Hypertension

Advanced glycation end products (AGEs) are implicated in the pathogenesis of vascular damage, especially in patients with diabetes and renal insufficiency. The oxidatively formed AGE N(epsilon)-carboxymethyllysine (CML) is thought to be a marker of oxidative stress. Four hundred fifty patients with type 2 diabetes and nephropathy from the Irbesartan in Diabetic Nephropathy Trial cohort (mean age, 58 +/- 8.2 years; 137 women, 313 men) with a mean glomerular filtration rate of 48.2 mL/min (0.80 mL/s; Modification of Diet in Renal Disease formula) were followed up for 2.6 years. Serum CML was measured by using an enzyme-linked immunosorbent assay. Relationships between CML levels, traditional risk factors, and cardiovascular and renal events were tested in Cox proportional hazards models. Mean serum CML level was 599.9 +/- 276.0 ng/mL, and mean hemoglobin A1c level was 7.5% +/- 1.6%. One hundred forty-three first cardiovascular events occurred during follow-up; 74 patients died, 44 of cardiovascular causes. Final multivariate analysis showed age (relative risk [RR], 1.87; confidence interval [CI], 1.13 to 3.11; P = 0.016 for the highest compared with lowest quartile), history of prior cardiovascular events (RR, 1.96; CI, 1.35 to 2.85; P < 0.0005), and 24-hour urinary albumin-creatinine ratio (RR, 1.29; CI, 1.11 to 1.50 per doubling; P < 0.0005) to be independent risk factors for a first cardiovascular event, but not CML level. CML level also did not correlate significantly with renal outcome. Serum CML level could not be identified as an independent risk factor for cardiovascular or renal outcomes in the examined population. This suggests that traditional risk factors might have a more important role for these end points or that other AGE compounds, as well as tissue AGE levels, might be of greater relevance compared with serum levels, which remains open to further study.

New therapies for advanced glycation end product nephrotoxicity: Current challenges

Background: The role of advanced glycation end products (AGEs) in diabetic nephropathy has been developed during several years of research and increasingly complex AGE biochemistry. However, the structural diversity of AGE chemistry has created new challenges in the search for AGE-based inhibition therapies. Results: The challenges include the need to standardize measurements of serum and tissue AGE levels, identifying nephrotoxic AGE compounds, understanding the cell biological state of AGEs in the diabetic kidney, determining the mechanism of action of selective inhibition of the glycation cascade, and forming complementary therapies. Conclusion: Current challenges in the development of new therapies for AGE nephrotoxicity are reviewed. Am J Kidney Dis 41(S1):S42-S47. © 2003 by the National Kidney Foundation, Inc.

Increased levels of advanced glycosylation end products in the kidney and liver from spontaneously diabetic Chinese hamsters determined by immunochemical assay

Increased levels of advanced glycosylation end products (AGEs) have been reported in tissues in association with diabetes mellitus. Thus, we measured tissue AGE levels and detected an accumulation of AGEs in the kidney and liver from spontaneously diabetic Chinese hamsters (CHAD) to determine the relationship between AGEs and diabetes mellitus. Diabetic CHAD aged 12 to 13 months were studied together with age-matched nondiabetic CHAD. We used an AGE-specific noncompetitive enzyme-linked immunosorbent assay (ELISA) with polyclonal anti-AGE-bovine serum albumin (BSA) antibody to measure tissue AGE levels. The samples extracted from the kidney and liver obtained from diabetic and nondiabetic CHAD reacted with anti-AGE-BSA antibody. When the absorbance of standard AGE-BSA (0.1 microg/mL) was expressed as 1 U, AGE levels in the kidney and liver from diabetic CHAD were significantly increased as compared with nondiabetic CHAD (kidney, 0.26 +/- 0.05 v 0.10 +/- 0.03 U/microg protein, P< .01; liver, 0.20 +/- 0.03 v 0.09 +/- 0.02 U/microg protein, P< .01). Positive AGE staining was observed in the renal cortex, especially in the tubules of diabetic CHAD, but little AGE staining was observed in the glomerulus by the immunohistochemical study. AGE staining was diffuse in the hepatocytes. These AGE levels were significantly correlated with fasting plasma glucose and glycated hemoglobin (P < .01, respectively). In conclusion, we have confirmed that AGE structures were expressed in the kidney and liver from CHAD, and these AGE levels were increased in diabetic CHAD. AGE staining was observed in the renal tubules and hepatocytes. Tissue AGE levels were positively correlated with glycemic control in CHAD.

Sleep apnoea in treated acromegaly: relative frequency and predisposing factors.

Sleep apnoea is common in active acromegaly. It is associated with increased morbidity and mortality but can be treated effectively. The objective of this study was to determine the largely unknown relative frequency of, and the predictive factors for, sleep apnoea in treated acromegalic patients. Retrospective cohort study. Tertiary referral hospital. Fifty-four of 100 patients with treated acromegaly. If sleep apnoea had been diagnosed before acromegaly, the patient was excluded. Sleep studies (using the MESAM-4 device measuring oxyhaemoglobin saturation, heart rate, snoring sounds and body position to determine presence and severity of sleep apnoea); GH and IGF-I levels; body mass index, neck and index-finger circumference; daytime symptoms of sleep apnoea, duration of acromegaly before treatment, shoe and neck-size difference since beginning of acromegaly; age, sex and treatment modes of acromegaly. The relative frequency of sleep apnoea was 39% in the 54 patients with sleep studies and at least 21% in the entire study population of 100 patients. In patients with sleep apnoea, statistically significant higher values were observed for GH (P = 0.002), IGF-I (P = 0.029), age (P = 0.014) and neck circumference (P = 0.016). An index-finger circumference of > or = 8.5 cm was associated with a significantly higher desaturation index (P = 0.012, Mann-Whitney U-test). Adenomectomy had been performed significantly less frequently in patients with sleep apnoea (P < 0.001, X2 test). The body mass index was non-significantly higher in the patients with sleep apnoea. The relative frequency of sleep apnoea in patients with treated acromegaly is at least 21%. Parameters of predictive value for the presence of sleep apnoea in this population are neck and index-finger circumference as measures of soft tissue hypertrophy, age, GH and IGF-I levels, and whether or not operative therapy was applied.

Formation of Immunochemical Advanced Glycosylation End Products Precedes and Correlates With Early Manifestations of Renal and Retinal Disease in Diabetes

Elevated levels of advanced glycosylation end products (AGEs) have been found in multiple tissues in association with diabetic vascular complications and during the microalbuminuric phase of diabetic nephropathy. In this study, we have used an AGE-specific enzyme-linked immunosorbent assay (ELISA) to measure skin AGEs to determine whether elevated levels can be detected before the onset of overt microangiopathy. Subjects with type I diabetes (n = 48) were graded for the degree of nephropathy (normal [23], microalbuminuria [12], or macroalbuminuria [12]) and retinopathy (none [13], background [20], or proliferative [15]). Subgroups with a premicroalbuminuric phase of albumin excretion (< or = 28 mg/24 h, n = 27) or with the earliest stages of retinopathy (n = 27) were identified. A significant increase in tissue AGEs was found as urinary albumin increased during the premicroalbuminuric phase of nephropathy even when the data were adjusted for age and duration of diabetes (P = 0.005). Immunoreactive AGEs also increased as normal renal status advanced to microalbuminuria and macroalbuminuria (P = 0.0001 across groups). Significant elevation of AGEs was also found in association with the earliest stages of clinically evident retinopathy (early background versus minimal grades). In addition, higher AGE levels were found in subjects with proliferative retinopathy when compared with those with less severe retinopathy (P < 0.004 across groups). In contrast, no significant differences were found in tissue AGE levels between groups with or without early retinopathy based on pentosidine or fluorescent AGE measurements, although fluorescent AGEs correlated with albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on leaves of Lachenalia

The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on Lachenalia leaf tissue were investigated. The genotypes studied, showed a variation in bud formation. The hybrid explants responded differently to factorial combinations of BA and NAA. The growth regulators could not substitute for the regeneration potential of the genotype. Tissue age had a pronounced effect on regeneration potential. Young tissue formed the largest number of buds. An interaction between tissue age and genotype was detected. Cytokinin levels in young leaf tissue were higher than in older tissue. In young tissue no relationship was observed between the cytokinin level and the number of buds formed. However, in older tissue it appears as if a relatively low endogenous cytokinin level enhanced bud formation.