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Technical Specification: Metabolism Assay Kits

Official Source Verification

This documentation is published by Beijing Solarbio Science & Technology Co., Ltd. For product procurement and commercial inquiries, visit the Solarbio Store.

1. Product Range

Assay SKU Detection Method Wavelength Detection Limit Linear Range Sample Types
ATP Content Assay Kit BC0300–BC0305 Hexokinase/G6PDH-coupled (phosphomolybdic acid) 636 nm 1 μmol/L 1–100 μmol/L Tissue, cells, bacteria, serum
Glucose Assay Kit (GOD-POD) BC2500 Glucose oxidase-peroxidase (Trinder) 505 nm 0.1 mmol/L 0.5–30 mmol/L Serum, plasma, culture media
Triglyceride Assay Kit BC0620 GPO-PAP enzymatic (lipoprotein lipase) 546 nm 0.05 mmol/L 0.1–10 mmol/L Serum, plasma, tissue
Total Cholesterol Assay Kit BC0650 CHOD-PAP enzymatic (cholesterol esterase) 505 nm 0.1 mmol/L 0.2–15 mmol/L Serum, plasma, tissue
Lactic Acid Assay Kit BC2230 Lactate oxidase-peroxidase colorimetric 530 nm 0.1 mmol/L 0.2–20 mmol/L Serum, plasma, tissue
Pyruvate Kinase Assay Kit BC2800 Kinetic NADH-linked (PEP → pyruvate) 340 nm (↓A) 1 U/L 3–300 U/L Tissue, cell lysate
Hexokinase (HK) Assay Kit BC0740 G6PDH-coupled (NADPH production) 340 nm (↑A) 0.2 U/L 0.5–100 U/L Tissue, cell lysate
Phosphofructokinase (PFK) Assay Kit BC1270 Fru-6-P → Fru-1,6-P₂ (NADH-linked) 340 nm (↓A) 0.5 U/L 1–100 U/L Tissue, cell lysate
PEPCK Assay Kit BC2170 P-enolpyruvate + GDP → GTP + OAA 340 nm (↑A) 1 U/L 2–200 U/L Liver tissue
G6Pase Assay Kit BC0745 Glucose-6-P → Glucose + Pi (colorimetric) 660 nm 0.5 U/L 1–100 U/L Liver, kidney
β-Hydroxybutyrate Assay Kit BC0460 β-HBDH kinetic (NADH production) 340 nm (↑A) 5 μmol/L 10–500 μmol/L Serum, plasma
Free Fatty Acid (NEFA) Assay Kit BC0590 ACS-ACOD-TOOS colorimetric 546 nm 0.02 mmol/L 0.05–2 mmol/L Serum, plasma, tissue

2. Detailed Assay Principles

2.1 ATP Content — Hexokinase/G6PDH Coupled with Phosphomolybdic Acid Detection

ATP content is a direct indicator of cellular energy status. The assay couples ATP-dependent glucose phosphorylation by hexokinase.

ATP + Glucose ──[HK]──→ Glucose-6-phosphate + ADP
G-6-P + NADP⁺ ──[G6PDH]──→ 6-Phosphogluconate + NADPH + H⁺
NADPH + PMS ──[Reduction]──→ Reduced PMS + NADP⁺
Reduced PMS + Phosphomolybdic acid → Molybdenum blue (636 nm)
Parameter Specification
Assay type Endpoint colorimetric
Wavelength 636 nm
Limit of detection 1 μmol/L ATP
Lower limit of quantification 2 μmol/L
Linear range 1–100 μmol/L ATP
Intra-assay CV <5% (n=20, 20 μmol/L)
Inter-assay CV <10% (n=8 lots)
Recovery rate 90–110%
Reaction time 30 min at 37°C
Sample volume 100 μL
Standard 100 μmol/L ATP (included)

Sample Preparation:

Sample Type Procedure Expected ATP (nmol/mg prot)
Liver tissue 0.1 g + 1 mL 0.6 M PCA, homogenize, centrifuge, neutralize 5–15
Muscle tissue 0.1 g + 1 mL 0.6 M PCA, bead mill homogenization 20–40
Cultured cells 1×10⁶ cells, lyse in 200 μL PCA, centrifuge, neutralize 5–20
Bacteria 1×10⁸ cells, boil in TE buffer 5 min, centrifuge 0.5–5

Critical Note: Perchloric acid (PCA) extraction must be neutralized with K₂CO₃/KOH to pH 7.0–7.5 before assay. Residual PCA denatures the coupling enzymes.

2.2 Glucose — GOD-POD (Trinder) Method

Glucose oxidase specifically oxidizes β-D-glucose to gluconic acid and H₂O₂. Peroxidase then couples H₂O₂ with 4-aminoantipyrine (4-AAP) and phenol to form a pink quinoneimine dye.

β-D-Glucose + O₂ + H₂O ──[Glucose Oxidase, pH 7.0, 37°C]──→ Gluconic acid + H₂O₂
2 H₂O₂ + 4-AAP + Phenol ──[POD]──→ Quinoneimine (pink, 505 nm) + 4 H₂O
Parameter Specification
Assay type Endpoint colorimetric
Wavelength 505 nm
Limit of detection 0.1 mmol/L
Linear range 0.5–30 mmol/L
Intra-assay CV <3% (n=20, 5 mmol/L)
Inter-assay CV <6% (n=8 lots)
Recovery 95–105%
Reaction time 15 min at 37°C
Sample volume 10 μL
Capacity 500 assays

Reference Values:

Sample Normal Fasting (mmol/L) Postprandial (mmol/L)
Human serum 3.9–6.1 <11.1 (2 h)
Mouse serum 4–10 —
Rat serum 4–8 —
Cell culture media 5 (DMEM) to 25 (high-glucose DMEM) —

Interference: Ascorbic acid (>0.5 mmol/L) and bilirubin (>200 μmol/L) lower apparent glucose concentration. For culture media, dilute 1:2–1:10 with saline to stay within linear range.

2.3 Triglyceride — GPO-PAP Method

Lipoprotein lipase (LPL) hydrolyzes triglycerides to glycerol and free fatty acids. Glycerol is then sequentially converted through coupled enzymatic reactions.

Triglyceride ──[LPL, 37°C]──→ Glycerol + 3 × Free fatty acids
Glycerol + ATP ──[GK]──→ Glycerol-3-phosphate + ADP
G-3-P + O₂ ──[GPO]──→ Dihydroxyacetone phosphate + H₂O₂
H₂O₂ + 4-AAP + 3,5-DHBS ──[POD]──→ Quinoneimine (red, 546 nm)
Parameter Specification
Assay type Endpoint colorimetric
Wavelength 546 nm
Limit of detection 0.05 mmol/L
Linear range 0.1–10 mmol/L
Intra-assay CV <4%
Inter-assay CV <8%
Recovery 92–108%
Sample volume 10 μL
Reaction time 10 min at 37°C
Free glycerol blank Included (subtract free glycerol for accurate TG)

Reference Values:

Species Normal (mmol/L) High (>mmol/L)
Human 0.45–1.69 >2.26
Mouse (C57BL/6) 0.5–1.2 >1.5 (high-fat diet)
Rat 0.4–1.0 —

Critical Note: For tissue homogenates, free glycerol blank subtraction is essential. The kit includes a separate blank reagent without LPL.

2.4 Total Cholesterol — CHOD-PAP Method

Cholesterol esters are hydrolyzed by cholesterol esterase (CE), and free cholesterol is oxidized by cholesterol oxidase (CO).

Cholesterol ester + H₂O ──[CE, 37°C]──→ Free cholesterol + Fatty acid
Free cholesterol + O₂ ──[CO]──→ Cholest-4-en-3-one + H₂O₂
2 H₂O₂ + 4-AAP + Phenol ──[POD]──→ Quinoneimine (505 nm)
Parameter Specification
Assay type Endpoint colorimetric
Wavelength 505 nm
Limit of detection 0.1 mmol/L
Linear range 0.2–15 mmol/L
Intra-assay CV <3%
Inter-assay CV <6%
Sample volume 10 μL
Reaction time 10 min at 37°C

Reference Values:

Species Normal (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L)
Human 3.1–5.7 0.9–1.6 <3.4
Mouse (C57BL/6) 1.5–3.0 1.2–2.5 0.2–0.5
Rat 1.5–2.5 0.8–1.5 0.3–0.6

2.5 Lactic Acid — Lactate Oxidase Method

Lactic acid (lactate) is a product of anaerobic glycolysis. The assay uses L-lactate oxidase to produce H₂O₂, detected colorimetrically.

L-Lactate + O₂ ──[Lactate Oxidase, pH 7.0]──→ Pyruvate + H₂O₂
2 H₂O₂ + 4-AAP + TOOS ──[POD]──→ Quinoneimine (purple, 530 nm)
Parameter Specification
Detection limit 0.1 mmol/L
Linear range 0.2–20 mmol/L
Intra-assay CV <4%
Sample types Serum, plasma, tissue homogenate, cell culture media
Reaction time 10 min at 37°C

Reference Values:

Sample Normal (mmol/L) Pathological
Human serum/plasma 0.5–2.2 >4 (lactic acidosis)
Mouse serum 1–4 Increases with exercise, ischemia
Cell culture supernatant 1–5 (confluent) 10–20 (hypoxia)
Tissue (muscle) 5–20 nmol/mg prot —

2.6 Pyruvate Kinase (PK)

Pyruvate kinase catalyzes the final step of glycolysis: phosphoenolpyruvate to pyruvate with ATP generation.

PEP + ADP ──[PK, 37°C]──→ Pyruvate + ATP
Pyruvate + NADH + H⁺ ──[LDH]──→ L-Lactate + NAD⁺
(↓A₃₄₀, rate proportional to PK activity)
Parameter Specification
Assay type Kinetic (NADH consumption)
Wavelength 340 nm
Limit of detection 1 U/L
Linear range 3–300 U/L
Intra-assay CV <6%
Unit definition 1 U = 1 μmol PEP converted per min

2.7 Hexokinase (HK)

Hexokinase catalyzes the first committed step of glycolysis: glucose phosphorylation.

Glucose + ATP ──[HK, 37°C]──→ Glucose-6-phosphate + ADP
G-6-P + NADP⁺ ──[G6PDH]──→ 6-Phosphogluconate + NADPH + H⁺
(↑A₃₄₀, rate proportional to HK activity)
Parameter Specification
Assay type Kinetic
Wavelength 340 nm
Detection limit 0.2 U/L
Linear range 0.5–100 U/L
Intra-assay CV <5%

2.8 Phosphofructokinase (PFK)

PFK catalyzes the rate-limiting step of glycolysis: fructose-6-phosphate to fructose-1,6-bisphosphate.

Fru-6-P + ATP ──[PFK, 37°C]──→ Fru-1,6-P₂ + ADP
Fru-1,6-P₂ ──[Aldolase]──→ G-3-P + DHAP
G-3-P ──[TPI]──→ DHAP (converted)
2 DHAP + 2 NADH ──[GDH]──→ 2 Glycerol-3-P + 2 NAD⁺
(↓A₃₄₀, 2 NADH consumed per Fru-1,6-P₂ formed)
Parameter Specification
Assay type Kinetic (2-step coupled)
Wavelength 340 nm
Detection limit 0.5 U/L
Linear range 1–100 U/L

2.9 PEPCK (Phosphoenolpyruvate Carboxykinase)

PEPCK is a key gluconeogenic enzyme that converts oxaloacetate to phosphoenolpyruvate.

OAA + GTP ──[PEPCK, 37°C]──→ PEP + GDP
PEP + ADP ──[PK]──→ Pyruvate + ATP
Pyruvate + NADH ──[LDH]──→ Lactate + NAD⁺
(↓A₃₄₀, rate proportional to PEPCK activity)
Parameter Specification
Detection limit 1 U/L
Linear range 2–200 U/L
Intra-assay CV <7%
Tissue Liver (highest), kidney, adipose

2.10 Glucose-6-Phosphatase (G6Pase)

G6Pase catalyzes the terminal step of gluconeogenesis and glycogenolysis. The generated phosphate is detected via molybdenum blue.

Glucose-6-P + H₂O ──[G6Pase, pH 6.5, 37°C]──→ Glucose + Pi
Pi + (NH₄)₆Mo₇O₂₄ + Reducing agent → Molybdenum blue (660 nm)
Parameter Specification
Assay type Endpoint (inorganic phosphate detection)
Wavelength 660 nm
Detection limit 0.5 U/L
Linear range 1–100 U/L

Critical Note: Microsomes must be intact for G6Pase activity. Freeze-thaw or detergents permeabilize microsomal membranes and may increase apparent activity (latency release).

2.11 β-Hydroxybutyrate (β-HB)

β-Hydroxybutyrate is the most abundant ketone body, elevated in ketosis, fasting, and diabetic ketoacidosis.

β-Hydroxybutyrate + NAD⁺ ──[β-HBDH, pH 9.5]──→ Acetoacetate + NADH + H⁺
(↑A₃₄₀, proportional to β-HB concentration)
Parameter Specification
Limit of detection 5 μmol/L
Linear range 10–500 μmol/L
Intra-assay CV <6%
Reference (human, fed) 10–300 μmol/L
Pathological >800 μmol/L (ketosis)

2.12 Free Fatty Acid (NEFA) — ACS-ACOD Method

Free fatty acids are activated by acyl-CoA synthetase (ACS), then oxidized by acyl-CoA oxidase (ACOD) producing H₂O₂.

FFA + ATP + CoA ──[ACS]──→ Acyl-CoA + AMP + PPi
Acyl-CoA + O₂ ──[ACOD]──→ 2,3-trans-Enoyl-CoA + H₂O₂
H₂O₂ + 4-AAP + TOOS ──[POD]──→ Quinoneimine (purple, 546 nm)
Parameter Specification
Limit of detection 0.02 mmol/L
Linear range 0.05–2 mmol/L
Intra-assay CV <5%
Reference (human serum) 0.1–0.9 mmol/L

3. Sample Preparation Guide

Assay Tissue Protocol Cell Protocol Serum/Plasma
ATP PCA extraction (0.6 M), neutralize to pH 7.0 PCA extraction, 1×10⁶ cells Direct assay possible (PCA needed for protein removal)
Glucose — — Direct; dilute culture media
Triglyceride Chloroform:MeOH (2:1) extraction Same as tissue Direct; free glycerol blank required
Cholesterol Chloroform:methanol extraction Same as tissue Direct
Lactic Acid 0.1 g in 0.5 mL saline, homogenize Lyse 1×10⁶ cells in 200 μL Direct; use fluoride/oxalate plasma
PK 0.1 g in 1 mL extraction buffer 1×10⁶ cells in 200 μL —
HK 0.1 g in 1 mL extraction buffer + 0.1% Triton 1×10⁶ cells in 200 μL —

4. Interference Table

Substance Glucose (GOD-POD) TG (GPO-PAP) Cholesterol (CHOD-PAP) Lactic Acid β-HB NEFA
Ascorbic acid (>0.5 mM) Strong Moderate Strong Moderate None Moderate
Bilirubin (>200 μmol/L) Moderate Moderate Moderate None None None
Hemoglobin (>1 g/L) Moderate Moderate Moderate Strong None Moderate
EDTA None None None None None None
Heparin None None None None None None
DTT (>1 mM) Strong Strong Strong Moderate None Strong

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